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INFLUENCE OF PROTEIN NUTRITION AND EXERCISE ON
MUSCLE METABOLISM
By
LEIGH BREEN, BSc, MSc.
A thesis submitted to
The School of Sport and Exercise Sciences
The University of Birmingham
For the Degree
DOCTOR OF PHILOSOPHY
School of Sport and Exercise Sciences
The University of Birmingham
October 2010
© Copyright by Leigh Breen
University of Birmingham Research Archive
e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
L. Breen: PhD Thesis II
ABSTRACT
The influence of protein nutrition on endurance performance has received growing
attention over the last several years. At present, there is no clear consensus as to whether
protein feeding harnesses any ergogenic benefit for endurance athletes, due to
methodological differences between the studies conducted to date. Whilst prior studies
suggest that endurance capacity may be extended with additional protein, chapters in this
thesis demonstrate no effect of protein on endurance performance. In addition to endurance
capacity, protein ingestion during and following exercise has been advocated to enhance the
recovery process. However, the impact of carbohydrate-protein co-ingestion on recovery
from intense exercise is considered to be somewhat equivocal, due to the practical relevance
of different tests of recovery and the lack of a clear physiological mechanism. Indeed, data
presented herein indicates that protein co-ingestion does not enhance recovery at 24 hours
post-exercise. Consequently, there is currently no basis on which to recommend protein
feeding for endurance performance and recovery.
Nutrient strategies implemented in the post-exercise period can markedly alter the acute
response of muscle protein synthesis. Thus, over an extended period of training appropriate
post-exercise nutrient manipulations may modulate metabolic adaptations. Whereas the
metabolic and molecular response to protein nutrition with resistance exercise has received
increasing attention, protein feeding following endurance exercise has received considerably
less. The combination of endurance exercise and protein ingestion increases the synthesis of
mixed muscle protein via the phosphorylation of signalling intermediates implicated in
mRNA translation initiation and elongation. Herein, we demonstrate that the response of
mixed muscle proteins to post-endurance exercise protein feeding is due, in large part, to an
increase in the synthesis of contractile proteins. Furthermore we provide evidence of altered
signalling phosphorylation with protein feeding which may explain the protein synthetic
response.
Insulin resistance that precedes Type II diabetes is characterized by blunted sensitivity
of the pancreas to glucose and impaired glucose uptake in skeletal muscle. It is suggested that
defective intracellular signalling, crucial for glucoregulation, may lead to hyperglycemia.
However, lifestyle interventions including nutrient manipulations and exercise have the
potential to improve glycemic control and can effectively restore glycemic control. Indeed, a
L. Breen: PhD Thesis III
single bout of resistance exercise can improve insulin sensitivity and glycemic control for up
to 48 hours post-exercise. In addition to resistance exercise, co-ingesting protein and/or
amino acids with a carbohydrate load facilitates the release of insulin, thereby suppressing the
prevailing plasma glucose response. The final experimental chapter in this thesis shows that
the rate of glucose disposal from the circulation is elevated 24 hours after a single bout of
resistance exercise in healthy normoglycemic males. It appears that prior exercise increased
the basal and insulin-stimulated phosphorylation of proximal intracellular signalling
intermediates regulating glucose uptake in skeletal muscle. Interestingly, protein ingestion
did not augment the glucose-lowering effects of prior resistance exercise.
KEYWORDS: Endurance cycling, muscle damage, muscle protein synthesis, resistance
exercise, glucose metabolism, intracellular signalling.
L. Breen: PhD Thesis IV
“Achievement is not always success while reputed failure often is. It is honest endeavour,
persistent effort to do the best possible under any and all circumstances”.
Orison Swett Marden, writer and physician (1850 - 1924)
L. Breen: PhD Thesis V
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere gratitude to Professors Kevin
Tipton and Asker Jeukendrup, for giving me the opportunity to pursue a PhD. Kevin, I would
like to thank you for the many lessons you have taught me and for helping shape my career in
science. Your thirst for knowledge and passion for research is inspirational and working
alongside you has heightened my enthusiasm for science. I thank you for the doors you have
opened for me and the many great scientists & friends you have introduced me to. Your
guidance means I will always owe you a debt of gratitude. I sorely miss your day-to-day
presence as a mentor and a great friend. That having been said, I hope we will have many
opportunities to work together (and share a few beers) in the future. Asker, the dedication and
work ethic you apply in balancing your many commitments has taught me that in order to
achieve success in my career I must endeavour to apply these same principles. I thank you for
your time, patience and advice. Additionally i would like to thank Drs Andrew Blannin and
Chris Shaw of the University of Birmingham and Professor Luc Van-Loon of Maastricht
University for taking time out of their busy schedules to serve on my PhD defence
committee.
I gratefully acknowledge the contribution of the world-leading collaborators I have
been fortunate enough to work alongside during my PhD. I hope through the time I have
spent working alongside all of you, we will have a long and successful working relationship
ahead of us. I thank the research team in The Division of Clinical Physiology, University of
Nottingham, for their expert analytical assistance and generosity in giving me ‘free reign’ in
their laboratory. To Drs Keith Baar and Andy Philp at The University of Davis, California,
for teaching me important analytical techniques that have strengthened my research and
helped me develop as an independent scientist. To Andy and Ash, I thank you for making a
fellow Brit welcome and keeping me entertained during my visits to Davis. I would like to
extend my gratitude to Professor Stu Phillips of McMaster University and Dr Dan Moore
(formerly of McMaster University) for allowing me to spend several weeks working in their
laboratory, learning a technique that has proved central to my thesis. To see a laboratory
working so efficiently and to such high standards was truly impressive. To my great friends
Dan and Beth, I thank you for your generosity in hosting me and showing me the pubs around
small town Ontario (every single one I think!).
L. Breen: PhD Thesis VI
Special thanks go to my fellow SportEx post-grads, both past and present, for ensuring
my social life in Birmingham was constantly active and my mind not always on work. I take
away with me some of the happiest memories of my life and many great friendships. I wish to
address my sincere thanks to my long-term housemates and fellow SportEx post-grads, Andy
and Stu, for always being there on days when there seemed like no light at the end of the
tunnel. You both are, and always will be, two of my closest friends. From a professional
perspective I extend my gratitude to the members of The Human Performance Laboratory I
have had the privilege to work alongside, particularly Ollie Witard and Sarah Jackman, who
have played a prominent role in assisting me during the completion of my research. The time
consuming and sometimes frustrating nature of metabolic research is made easier when such
dedicated colleagues are available to call on. Finally, I would like to pay tribute to those
underappreciated individuals who manage the day-to-day running of SportEx; Val Queeley,
Hazel Robinson, Steve Allen and Rob Wheeler (to name but a few) provide a fantastic
service to the school and their collective presence has helped me enormously during my time
in Birmingham.
To my wonderful family I dedicate this thesis. You have instilled in me the work ethic
necessary to complete this long (and often arduous) journey. I thank you for motivating me
during the tough times and for your financial help, which has kept my head above water
through the years. All of the early-mornings and the many hours spent in the lab were all to
make you proud; something I hope I have achieved. Last, but certainly not least, my most
heartfelt thanks go to the love of my life, Hannah, who has been by my side throughout my
university life. It is impossible for me to put into words just how much your encouragement,
patience and belief in my abilities means to me. Rest assured my heart will always be yours.
Finally, I wish to acknowledge GlaxoSmithKline Nutritional Healthcare and The
Insulin Dependent Diabetes Trust, who have financially supported the work presented in this
thesis. I would also like to acknowledge the contribution of all the participants who invested
their time, effort and muscle tissue for these experiments.
L. Breen: PhD Thesis VII
TABLE OF CONTENTS
Abstract II
Acknowledgments V
List of tables XIII
List of Figures XIV
Publications XVI
Chapter 1: General Introduction
1.1 Introduction 1
1.1.1 Molecular basis of exercise training adaptations 3
1.1.2 Protein metabolism in skeletal muscle tissue 4
1.1.3 Intracellular signalling regulators of protein metabolism 5
1.2 Influence of nutrition on muscle protein synthesis 6
1.2.1 Fate of dietary amino acids 7
1.2.2 Muscle protein synthesis after feeding 8
1.2.3 Synthetic response of different protein fractions after feeding 10
1.2.4 Intracellular signalling response to feeding 12
1.3 Metabolic and cellular adaptations to endurance exercise and nutrition 14
1.3.1 Endurance exercise adaptations 15
1.3.2 Muscle protein metabolism after endurance exercise 16
1.3.3 Intracellular signalling response to endurance exercise 18
1.3.4 Muscle protein synthesis after post-endurance exercise protein feeding 20
L. Breen: PhD Thesis VIII
1.3.5 Intracellular signalling to endurance exercise and protein feeding 21
1.4 Influence of protein nutrition on endurance performance and recovery 24
1.4.1 Carbohydrate-protein co-ingestion for endurance exercise performance 25
1.4.2 Carbohydrate-protein co-ingestion for indirect markers of muscle damage 27
1.5 Glucose metabolism in skeletal muscle 30
1.5.1 Intracellular regulation of glycemic control in skeletal muscle 30
1.5.2 Type II diabetes and insulin resistance 31
1.5.3 Mechanisms of insulin resistance in peripheral tissues 32
1.6 Influence of exercise on glucose homeostasis 33
1.6.1 Resistance exercise and glycemic control 34
1.6.2 Intracellular signalling regulating glycemic control with acute exercise 35
1.7 Influence of protein nutrition on glucose homeostasis 37
1.7.1 Insulin-stimulated hypoglycemia with protein co-ingestion 38
1.7.2 Protein absorption and hydrolysis for glucose homeostasis 39
1.7.3 Insulinotropic properties of free amino acids 40
1.7.4 Mechanisms of glucose uptake with amino acids 41
1.8 Specific objectives of the thesis 45
1.9 References 46
Chapter 2: No Effect of Carbohydrate-Protein on Cycling Performance and Indices of
Recovery.
2.1 Abstract 69
2.2 Introduction 71
L. Breen: PhD Thesis IX
2.3 Methods 73
2.3.1 Participants 73
2.3.2 Experimental design 73
2.3.3 Baseline testing 74
2.3.4 Blinded trials 76
2.3.5 Treatment beverages 78
2.3.6 Diet & exercise control 79
2.3.7 Statistical analysis 80
2.4 Results 81
2.4.1 Time-trial performance 81
2.4.2 Steady-state exercise 81
2.4.3 Isometric knee extensor strength 82
2.4.4 Visual analogue scale of muscle soreness 83
2.4.5 Plasma creatine kinase concentration 83
2.4.6 Treatment blinding 83
2.5 Discussion 86
2.6 References 92
Chapter 3: The influence of carbohydrate-protein co-ingestion following endurance
exercise on myofibrillar and mitochondrial protein synthesis.
3.1 Abstract 97
3.2 Introduction 99
3.3 Methods 101
L. Breen: PhD Thesis X
3.3.1 Participants 101
3.3.2 Study design 101
3.3.3 Preliminary testing 102
3.3.4 Experimental trial protocol 103
3.3.5 Analyses 106
3.3.6 Calculations 110
3.3.7 Statistical analysis 111
3.4 Results 111
3.4.1 Exercise variables 111
3.4.2 Blood analytes 112
3.4.3 Plasma amino acid concentrations 112
3.4.4 Plasma and intracellular phenylalanine enrichment 113
3.4.5 Post-exercise protein phosphorylation 113
3.4.6 Mitochondrial and myofibrillar FSR 114
3.5 Discussion 123
3.6 References 130
Chapter 4: Beneficial effects of resistance exercise on glycemic control are not further
improved with protein ingestion.
4.1 Abstract 136
4.2 Introduction 138
4.3 Methods 140
4.3.1 Participants 140
L. Breen: PhD Thesis XI
4.3.2 Study design 140
4.3.3 Preliminary testing 142
4.3.4 Treatment trials 143
4.3.5 Treatment beverages 145
4.3.6 Analyses 146
4.3.7 Calculations 148
4.3.8 Statistical analysis 149
4.4 Results 151
4.4.1 Dietary intake 151
4.4.2 Exercise variables 152
4.4.3 Plasma glucose 152
4.4.4 Plasma insulin 152
4.4.5 Glucose tracer kinetics 153
4.4.6 Muscle glycogen 154
4.4.7 Protein phosphorylation 154
4.5 Discussion 162
4.6 References 169
Chapter 5: General Discussion
5.1 Protein nutrition for endurance performance 176
5.1.1 Future studies 177
5.2 Muscle adaptation to acute endurance exercise and protein nutrition 178
5.2.1 Future studies 179
L. Breen: PhD Thesis XII
5.3 Protein nutrition for recovery from endurance exercise 181
5.3.1 Future studies 182
5.4 Metabolic adaptation to acute resistance exercise and protein nutrition 183
5.4.1 Future studies 184
5.5 Conclusions 187
5.6 References 188
Chapter 6: Appendices - Method development
6.1 Method of mitochondrial and myofibrillar protein isolation 193
6.1.1 Method preparation and supplies 194
6.1.2 Sample preparation prior to isolation 195
6.1.3 Reagent preparation 195
6.1.4 Analysis of 13C6 phenylalanine enrichment by gas chromatography-combustion-
isotope ratio mass spectrometry 198
6.1.5 Citrate synthase activity assay 199
6.1.6 Western blots 200
6.2 Method of plasma glucose enrichment 202
6.2.1 Reagent preparation 202
6.2.1 Protocol for deproteinisation and derivitization of samples 202
6.2.3 Analysis of [U-13
C] and 6, 6-[2H2] glucose enrichment by gas
chromatography-mass spectrometry 203
6.3 References 203
L. Breen: PhD Thesis XIII
LIST OF TABLES
Chapter 1
Table 1.1 Comparison of studies examining muscle protein synthesis and intracellular
signalling following endurance exercise. 23
Table 1.2 Comparison of studies examining endurance performance and recovery
following carbohydrate-protein feeding. 29
Table 1.3 Comparison of studies examining glucose metabolism and intracellular signalling
in response to protein/amino acids on in rat and cell culture models. 44
Chapter 2
Table 2.1 Participants characteristics at baseline. 74
Table 2.2 Comparison of habitual and standardized diet. 82
Table 2.3 Physiological data at 25, 50, 75 and 100% of time-trial completion. 82
Chapter 3
Table 3.1 Exercise variables. 116
Table 3.2 Post-exercise protein phosphorylation. 120
Chapter 4
Table 4.1 Participant characteristics. 145
Table 4.2 Participant dietary composition. 155
Table 4.3 Plasma glucose kinetics. 160
L. Breen: PhD Thesis XIV
LIST OF FIGURES
Chapter 1
Figure 1.1 Simplified schematic representation of the intracellular signalling regulation of
muscle protein synthesis. 12
Figure 1.2 Insulin-, BCAA- and contraction-stimulated signalling mechanisms of glucose
uptake in skeletal muscle. 43
Chapter 2
Figure 2.1 Schematic diagram of the experimental protocol. 79
Figure 2.2 Average power output measured at quarterly completion of time-trial target 84
Figure 2.3 Time-trial time-to-completion. 84
Figure 2.4 Maximum isometric force production of the knee extensors at baseline and 24 h
post-exercise. 85
Figure 2.5 Average visual analogue scale of perceived muscle soreness at baseline and 24 h
post-exercise. 85
Figure 2.6 Plasma creatine kinase concentrations at rest and 24 h post-exercise. 86
Chapter 3
Figure 3.1 Schematic diagram of the experimental protocol. 106
Figure 3.2 Plasma urea (A) and serum insulin (B) concentration. 117
Figure 3.3 Plasma concentrations of phenylalanine (A), leucine (B) and threonine (C). 118
Figure 3.4 Enrichment of 13
C6 phenylalanine in plasma. 119
L. Breen: PhD Thesis XV
Figure 3.5 Post-exercise protein phosphorylation of mTORSer2448
(A), eEF2Thr56
(B) and
p70S6KThr389
(C). 121
Figure 3.6 Myofibrillar and mitochondrial fractional synthetic rate. 123
Chapter 4
Figure 4.1 Schematic diagram of the study protocol. 150
Figure 4.2 Plasma glucose concentration and AUC. 161
Figure 4.3 Plasma insulin concentration and AUC. 162
Figure 4.4 Tracer enrichment and glucose kinetics during OGTT. 163
Figure 4.5 Muscle glycogen content. 165
Figure 4.6 Representative protein phosphorylation blots 165
Figure 4.7 Protein phosphorylation. 166
Chapter 5
Figure 5.1 Proposed time-course of net myofibrillar protein balance following endurance
exercise and protein feeding. 181
Chapter 6
Figure 6.1 Protein isolation protocol. 197
Figure 6.2 Citrate synthase activity assay. 200
Figure 6.3 Western blot analysis of isolated protein fraction purity. 201
L. Breen: PhD Thesis XVI
LIST OF PUBLICATIONS
Published
L. Breen., K. D. Tipton and A. E. Jeukendrup. No effect of carbohydrate-protein on cycling
performance and indices of recovery. Med Sci Sports Ex. 2010; 42(6) 1140-1148.
Submitted
L. Breen., C. S. Shaw., K. D. Tipton and A. E. Jeukendrup. Understanding the role of protein
and amino acid nutrition in the treatment of Type II diabetes (In review).
L. Breen., A. Philp., O. C. Witard., S. R. Jackman., A. Selby., K. Smith., M. J. Rennie., K.
Baar and K. D. Tipton. The influence of carbohydrate-protein co-ingestion following
endurance exercise on myofibrillar and mitochondrial protein synthesis (in review).
In preparation
L. Breen., A. Philp., C. S. Shaw., A. E. Jeukendrup., K. Baar and K. D. Tipton. Beneficial
effects of resistance exercise on glycemic control are not further improved by protein
ingestion.
L. Breen., A. Philp., K. Baar and K. D. Tipton. Review: The physiological relevance of
muscle protein synthesis with aerobic exercise.
Abstracts
L. Breen: PhD Thesis XVII
L. Breen, K. D. Tipton and A. E. Jeukendrup. Can the addition of protein to a carbohydrate
beverage improve endurance performance and enhance recovery? Proceedings of the 13th
annual congress of the European College of Sports Science 2008.
L. Breen., A. Philp., C. S. Shaw., A. E. Jeukendrup., K. Baar and K. D. Tipton. Beneficial
effects of resistance exercise on glycemic control are not further improved by protein
ingestion. Proceedings of the 58th
annual American College of Sports Medicine meeting
2011.
General Introduction 1
CHAPTER 1
GENERAL INTRODUCTION
1.1 Introduction
The quality and quantity of nutritional intake harnesses many benefits for athletic
populations. Nutritional strategies implemented prior to and during exercise can profoundly
alter exercise performance and prolong endurance capacity. Further, effective post-exercise
nutritional strategies can replenish depleted energy stores, attenuate indices of muscle
damage and augment the recovery of endurance capacity and muscle function. In addition to
exercise performance and recovery, dietary modifications can profoundly alter the adaptive
response to exercise. Although the major perturbations to cellular homeostasis and muscle
substrate stores occur during exercise, the activation of intracellular signalling pathways
important for chronic training adaptations are thought to occur during the first few hours of
recovery, (114, 209). This has led to suggestions that many chronic training adaptations are
generated by the cumulative effects of the transient events that occur during recovery from
each (acute) exercise bout. Evidence is accumulating to suggest that nutrient intake can serve
as a potent modulator of many of the acute responses to exercise training.
Protein, derived from the Greek word proteos, means primary or ‘most important’. In
the sporting environment, protein nutrition was initially favoured by bodybuilders, but it now
a common consideration for the majority of athletes. However, the effectiveness of protein
nutrition for endurance athletes is yet to be fully elucidated. In combination with
methodological limitations that cloud our understanding of how protein feeding can impact
General Introduction 2
endurance performance and recovery, very little is known regarding the influence of protein
nutrition on the metabolic and molecular adaptations to endurance exercise. Thus, improving
our understanding of how nutritional strategies can optimize the adaptive response to exercise
could provide the knowledge necessary to allow athletes to successfully modify their training
and dietary habits and achieve their competitive goals. In light of evidence to suggest that
protein nutrition could be important for endurance athletes, chapters in this thesis detail
studies that have focussed on determining the effects of protein nutrition on endurance
performance, indices of recovery and metabolic and cellular adaptations to endurance
exercise.
Beyond athletic performance, lifestyle interventions such as exercise training and
dietary manipulations have the potential to prevent or delay the incidence of metabolic
disorders. Skeletal muscle mass accounts for ~50% of total body mass. Accordingly, skeletal
muscle is a substantial contributor to basal metabolic rate (135). Furthermore, skeletal muscle
represents the prime storage depot for body amino acids and is a critical metabolic tissue
involved in glucose disposal and lipid oxidation, particularly during periods of exercise (2,
176, 274). As such, maintaining a large and metabolically active skeletal muscle mass
through exercise, has some influence on the risk for metabolic disorders (e.g., Type II
diabetes) as well as all-cause mortality in a variety of diseased states (274). Indeed it is
known that a single bout of resistance exercise can improve glucose tolerance and insulin
sensitivity for up to 48 hours post-exercise in humans (176, 205). At the level of skeletal
muscle, the mechanisms facilitating this response are not fully understood.
In addition to contractile stimuli, nutritional modifications can be used to fulfil several
distinct roles in the treatment of metabolic disorders, for example, by increasing weight-loss
General Introduction 3
and lowering glucose concentrations. Besides the application of energy restricted diets, it has
been suggested that specific food components can be applied to directly modulate glycemic
control. In this regard, cell culture and animal studies elegantly show that protein and/or
branched chain amino acids can promote skeletal muscle glucose uptake (and removal from
the circulation) via the activation of insulin-dependent and insulin-independent signalling
pathways. In the absence of clear evidence in humans, that exercise and protein nutrition can
improve metabolic health parameters, chapters in this thesis will also focus on examining the
response of glucose metabolism to an acute bout of resistance exercise-alone or resistance
exercise with protein feeding.
1.1.1 Metabolic basis of exercise training adaptations
Skeletal muscle is a malleable tissue capable of altering the type and amount of protein
in response to disruptions to cellular homeostasis. The complex process of exercise-induced
adaptation in skeletal muscle involves specific signalling mechanisms initiating replication of
DNA genetic sequences (151) that enable subsequent translation of the genetic code into a
series of amino acids to create new proteins (31). Skeletal muscles sense and distinguish
contraction-specific signals to produce adaptations over time that are specific to the nature,
intensity and duration of exercise (129, 167). However, it is thought that there is substantial
overlap in the molecular response to different exercise modes (156). Contraction generates
transient increases in the quantity of messenger RNA (mRNA) that for a multitude of genes
typically peaks within 12 hours post-exercise and returns to basal levels within 24 hours (21,
209, 275). This directional change in mRNA is generally the same as the encoded protein
during adaptation to a new steady-state level (30). Thus, contractile stimuli acutely increase
transcriptional activity and the subsequent synthesis of new proteins and may be potentiated
when appropriate nutrient strategies are implemented post-exercise. In summary, chronic
General Introduction 4
adaptations to training are thought to represent the summation of each discrete exercise bout,
with these perturbations leading to cumulative alterations in gene and protein expression and
ultimately distinct phenotypic changes (112, 167).
However, the ability to infer phenotypic changes in skeletal muscle from ‘snapshots’ of
gene expression is complicated by the fact that multiple factors may be involved in the
translation of mRNA species that can ultimately affect the expression of their protein
products such as, for example, the post-transcriptional modification or stability (via action of
chaperone proteins and RNAses) of mRNA transcripts and/or their translational regulation by
other molecules such as microRNAs (271). In the absence of measured changes in protein
abundance and/or the direct synthesis of their protein product it may be difficult to
conclusively ascertain the functional outcome of changes in gene transcription.
1.1.2 Protein metabolism in skeletal muscle tissue
Skeletal muscle proteins are constantly and simultaneously synthesized and degraded.
Net protein balance is defined as the difference between muscle protein synthesis and muscle
protein breakdown. Thus, a significant rise in skeletal muscle protein synthesis (anabolism)
and/or a reduction in muscle protein breakdown (catabolism), such that net protein balance
remains positive can result in the accretion of skeletal muscle proteins. Conversely, a
negative net protein balance, arising from a reduction in synthesis and/or increase in
breakdown, will result in a loss of skeletal muscle proteins. Net protein balance is maintained
by ingestion of protein-containing meals which results in systemic hyperaminoacidemia that
is stimulatory for the synthesis of new proteins (18, 27, 28, 83). Therefore, in persons with
consistent dietary habits who are not performing exercise, skeletal muscle protein mass
remains relatively stable. However, the feeding-induced stimulation of muscle protein
General Introduction 5
synthesis is only transient and even in the face of available amino acids returns to basal levels
(28). Exercise stimuli, aerobic and more so resistive, improve net protein balance. However,
the ingestion of protein during post-exercise recovery is necessary to shift net protein balance
in favour of muscle protein accumulation (23, 218). Thus, feeding and exercise must be
utilized concurrently to manifest a positive net protein balance and increase protein mass
when performed habitually.
In healthy individuals, changes in the rate of muscle protein synthesis are thought to
represent the primary locus of control for regulating overall muscle protein balance (218).
Evidence to support this contention is based on the following observations: i) the transition
from the fasted to the fed state is associated with a 60-80% stimulation of muscle protein
synthesis with no measurable change in breakdown (23, 260, 262); ii) the acute (i.e. 3 hours)
feeding and exercise-induced increase in net protein balance is due primarily to changes in
muscle protein synthesis and is additive to the daily net protein balance (242); iii) the acute
change in muscle protein synthesis is qualitatively predictive of long term changes in muscle
mass in response to both hypertrophic (i.e., resistance training) (82, 110, 116, 157, 268) and
atrophic (i.e., immobilization) stimuli (94).
1.1.3 Intracellular signalling mechanisms regulating protein metabolism
Acute changes in muscle protein synthesis are regulated at the level of mRNA
translation of which the primary rate controlling step is the initiation and formation of a
translation-competent ribosome (147). The first stage of mRNA translation involves charging
of initiator methionyl-tRNA, which is catalyzed by the guanine nucleotide exchange activity
of the ε-subunit of eukaryotic initiation factor (eIF) 2B (eIF2Bε) and its subsequent
General Introduction 6
association with eIF2. This ternary structure then binds to the 40S ribosomal subunit to form
the 43S pre-initiation complex. Following this, hyperphosphorylation of the negative
regulator 4E-binding protein 1 (4E-BP1) results in its release of eIF4E, which is then
permitted to bind to the mRNA and form the heterotrimeric eIF4F-mRNA complex. The
eIF4F-mRNA complex then combines with the 43S pre-initiation complex to form the 48S
pre-initiation complex, which subsequently binds to the 60S ribosomal subunit to form a
translationally competent ribosome. Although initiation is the primary regulated step in
mRNA translation, peptide chain elongation consumes the vast majority of energy during
protein synthesis and also represents another level of control for the synthesis of proteins in
vivo (263). The translocation of the ribosome along mRNA is catalyzed by eukaryotic
elongation factor 2 (eEF2), which is negatively regulated by phosphorylation that is catalyzed
by eEF2 kinase. Of particular importance to the regulation of protein synthesis appears to be
the mTOR-mediated activation of p70S6K, which phosphorylates both eIF4B, to enhance the
helicase activity of eIF4F complex and eEF2 kinase. Furthermore, hyperphosphorylation of
p70S6K and downstream target ribosomal protein (rp) S6 facilitates translation initiation by
increasing the affinity of ribosomes for binding 5’terminal oligopyridimine (5’TOP) mRNAs
(131). Thus, p70S6K plays a role in both the initiation and elongation stages of mRNA
translation (213). The signalling mechanisms regulating skeletal muscle protein synthesis are
presented in Figure 1.1.
1.2 Influence of nutrition on muscle protein synthesis
Skeletal muscle is a heterogeneous tissue, composed of a variety of different proteins
with distinct functions. From the standpoint of overall muscle mass and quality, the most
important protein fraction within skeletal muscle is myofibrillar, which is composed
General Introduction 7
primarily of the contractile proteins actin and myosin. However, skeletal muscle contains
many non-myofibrillar proteins and organelles (e.g., mitochondria, sarco- and endoplasmic
reticula, ribosomal proteins, etc.) that are found within the cytoplasm of the muscle cell and
therefore are collectively referred to as sarcoplasmic proteins. Mitochondrial proteins are
involved in energy production and sarcoplasmic proteins contribute to anaerobic energy
production, intracellular transport and numerous other functions. In addition, the external
support lattice of muscle cells is comprised primarily of both fibrillar and non-fibrillar
collagen protein. The majority of studies investigating the effects of nutrients on muscle
protein synthesis measure changes in a mixed muscle protein fraction. This practice has
provided the framework for our basic knowledge of how muscle responds to feeding but has
the obvious disadvantage of representing an average synthetic rate of all muscle proteins.
Measurement of the mixed muscle protein synthetic rate could mask potentially important
physiological differences within specific muscle protein fractions. Therefore, the following
sections of this chapter will discuss the changes in the synthesis of mixed muscle and specific
muscle fractions in response to feeding.
1.2.1 Fate of dietary amino acids
A substantial portion (up to ~80% for some) of dietary amino acids absorbed by the
small intestines, are subsequently catabolized or utilized for the synthesis of constitutive
hepatic and secreted proteins within gut tissues, with the balance being transported to the
liver to encounter a similar metabolic fate (174, 216). In addition to the synthesis of
constitutive hepatic and exported proteins (especially albumin (61)), dietary amino acids can
also be converted into glucose by the liver to provide a source of energy for other body
tissues such as, for example, the brain. Because of its high turnover rate and large metabolic
capacity, this first-pass splanchnic extraction, which includes both gut and liver metabolism,
General Introduction 8
represents a means for contending with large fluctuations in peripheral blood amino acid
concentrations following ingestion of large protein containing meals (174). However, in order
to support the synthesis of other body proteins there must be a net efflux of amino acids from
the liver into the peripheral circulation. These amino acids bypass first-pass extraction and
provide the substrates for the synthesis of a wide variety of body proteins, arguably the most
important of which, based on their role in locomotion and amino acid storage as well as their
collective contribution to whole body substrate metabolism, are skeletal muscle proteins.
Dietary amino acids transported from the circulation into the skeletal muscle enter the free
pool of amino acids whereby they can stimulate and support the synthesis of new muscle
proteins. In addition to their roles as substrates for muscle protein synthesis, amino acids can
also be deaminated and converted into a source of metabolic fuel for muscle and other tissues
of the body. This process occurs either directly via the production of tricarboxylic acid (TCA)
cycle intermediates, or indirectly via the production of precursors for hepatic
gluconeogenesis. Under conditions in which the delivery of dietary amino acids to the muscle
and other body tissue exceeds their ability to be incorporated into new proteins there is a
marked increase in amino acid catabolism (179). Therefore, it is apparent that, in addition to
the well defined role of stimulating and supporting muscle protein synthesis, the disposal of
dietary amino acids can also occur through other metabolic pathways within different tissues
of the body.
1.2.2 Muscle protein synthesis after feeding
It has been well established that the transition from a fasted to fed state is characterized
by the inward transport of amino acids into skeletal muscle and a marked increase in the
synthesis of muscle proteins (22, 217). The feeding-induced stimulation of protein synthesis,
which is consistent across different muscles and fibre types (41, 180), functions to restore the
General Introduction 9
obligatory losses of muscle protein that occur during the fasting so that on a day-to-day basis
muscle mass is maintained. The primary stimulatory effect of feeding arises almost
exclusively from the presence of the essential amino acids (236, 244, 261), in particular the
branched chain amino acid leucine (235). There is evidence to indicate that insulin can
enhance the synthesis of muscle proteins when administered alone (89) or in the presence of a
sub-optimal level of amino acids (i.e., a level below which protein synthesis is maximally
stimulated) (99). The mechanism by which insulin facilitates the protein synthetic response in
this situation is not direct but likely occurs via the enhanced delivery of nutrients to the
muscle, secondary to an insulin-induced vasodilation and capillary recruitment (51, 85). With
the provision of an optimal level of exogenous amino acids it appears that only basal insulin
concentrations are required to allow the full stimulation of muscle protein synthesis (95, 99).
These data suggest the role of insulin in healthy individuals is permissive rather than
stimulatory for the anabolic effect of amino acids. However, in the absence of amino acids
the role of post-prandial insulin concentrations for muscle anabolism may be more important
(89). Thus, the meal-induced stimulation of muscle protein synthesis is primarily regulated by
the presence of dietary amino acids.
Muscle protein synthesis is rapidly (i.e. within 30-90 min) increased in response to a
physiological increase in plasma amino acids (10, 28, 83). Interestingly, the stimulation of
muscle protein synthesis during a constant infusion of amino acids subsides after ~3 hours
despite the presence of a persistent hyperaminoacidemia (28). The presence of a refractory
period for protein synthesis (28) combined with the observation that the synthesis of mixed
muscle proteins is regulated by the extracellular rather than intracellular concentration of
amino acids (27), suggests that muscle protein synthesis may be stimulated primarily by the
acute change in plasma amino acid concentration in response to amino acid ingestion.
General Introduction 10
Subsequent studies have established that mixed muscle protein synthesis is rapidly stimulated
after the ingestion of dietary amino acids (83). Furthermore, recent studies have begun to
describe the temporal changes in the synthesis of specific muscle protein fractions after the
ingestion of physiological protein ingestion (10, 181) and intravenous infusion of mixed
amino acids (28).
1.2.3 Synthetic response of different protein fractions after feeding
It is thought that myofibrillar proteins are the prime storage depot for amino acids in the
body. This notion is supported by evidence that during periods of reduced amino acid
availability, myofibrillar proteins are preferentially targeted for degradation (272). Thus, in
order to maintain muscle mass, protein feeding must stimulate the synthesis of myofibrillar
proteins to counteract fasted-state losses. In spite of the critical role myofibrillar proteins play
in amino acid storage, few studies have examined the response of myofibrillar proteins to
alterations in nutrient status. Work by Bohé and colleagues (27, 28) showed that myofibrillar
proteins display similar sensitivity to a mixed muscle protein fraction during the provision of
amino acids, with regard to the magnitude and duration of synthesis. This is not surprising
considering that myofibrillar protein accounts for ~60-65% of all skeletal muscle protein and
would contribute substantially to rates of mixed muscle protein synthesis. However, it should
be noted that early studies examining the response of myofibrillar protein synthesis to protein
feeding infused amino acids intravenously, an approach which may not adequately describe a
physiological situation in which amino acids are ingested orally. More recent studies
demonstrate a similar amplitude and duration of myofibrillar and sarcoplasmic protein
synthesis in response to physiological whey protein ingestion (10, 181). These data are in
contrast to previous studies reporting that sarcoplasmic proteins are less sensitive to nutrient
availability than myofibrillar proteins (59, 180). Accepting that muscle protein synthesis is
General Introduction 11
regulated by acute changes in extracellular amino acid concentration (27), it is possible that a
bolus ingestion of 25g (181) and 48g (10) of protein supplied in these studies, enhanced the
sensitivity of sarcoplasmic proteins to nutrients, as a result of a greater physiological increase
in extracellular amino acid concentration compared with oral ingestion of 10g of crystalline
amino acids (59), or a sustained infusion of amino acids (180).
Sarcoplasmic proteins, in which mitochondrial proteins are included, are thought to be
less sensitive to nutrient availability than myofibrillar proteins (59, 180). Differences in the
nutrient sensitivity of the two protein fractions has been hypothesized to represent a
mechanism muscle composition to ensure that and the relative amount of myofibrillar and
sarcoplasmic proteins are maintained, despite differences in the rates of turnover (14, 59,
180). Due to technical limitations, changes in mitochondrial protein synthesis rates in
response to the ingestion of a physiological dose of protein have not been well characterized.
However, Bohé et al. (27, 28) showed the synthetic response of myofibrillar, sarcoplasmic
and mitochondrial proteins were similar in magnitude and duration following an amino acid
infusion. In union with the findings of Moore et al. (181), these data imply that an accelerated
increase in plasma amino acid concentration, typically found after the ingestion of rapidly
digested protein such as whey, might maximize the synthesis of sarcoplasmic and
mitochondrial proteins to match that of the myofibrillar fraction. Thus, to advance our
understanding of how non-myofibrillar proteins respond to nutrients it would be valuable to
characterize changes in the synthesis of mitochondrial muscle proteins to physiological
nutrient ingestion. Further to this suggestion, Moore et al. (181) recently showed the
combined effect of resistance exercise and whey protein feeding potentiated the synthetic
response of myofibrillar proteins. Thus, the synergistic effect of nutrient and contractile
General Introduction 12
Figure 1.1 A simplified schematic representation of the intracellular signalling mechanisms
controlling skeletal muscle protein synthesis by amino acids and insulin. Proteins have been
labelled to designate them as positive (green) or negative (red) regulators of mTOR and muscle
protein synthesis.
stimuli may be important for the synthesis of specific protein fractions and remains to be
fully elucidated.
1.2.4 Intracellular signalling response to feeding
Considerable advances have been made toward improving our understanding of the
intracellular signalling events that contribute to the activation and regulation of mRNA
translation in skeletal muscle. Of critical importance to the activation of muscle protein
General Introduction 13
synthesis with nutrient ingestion appears to be proteins within the mTOR signalling cascade.
For example, the feeding-induced increase in human skeletal muscle protein synthesis is
accompanied by an increase in the phosphorylation of mTOR and its downstream effectors
involved in mRNA translation initiation (p70S6K, 4E-BP1) and elongation (eEF2) (10, 60,
83, 93, 103, 234). The association between these signals and muscle protein synthesis was
recently brought to light by Atherton and colleagues (10), who demonstrated that the
‘upswing’ in muscle protein synthesis following amino acid ingestion is echoed in the
phosphorylation of several intermediates in the mTOR signalling pathway.
Although studies investigating the effect of amino acid feeding on intracellular
signalling in humans typically provide additional carbohydrates that would enhance insulin-
stimulated phosphorylation of mTOR by its upstream kinase Akt (83, 99), amino acids have
been shown to activate mTOR in vivo directly with only a basal requirement for insulin (105,
119, 263). It has recently been shown that during the constant provision of amino acids,
signalling molecule phosphorylation upstream and downstream of mTOR is enhanced by the
presence of insulin (73, 119). However, despite the incremental activation (evidenced by
greater phosphorylation) of signalling proteins in the mTOR cascade with graded insulin
concentration, there was no concomitant elevation of muscle protein synthesis (119). These
data suggest that a minimum level of phosphorylation is required to activate the signalling
proteins involved in the regulation of mRNA translation with nutrients, after which,
additional activation of the mTOR pathway, such as with hyperinsulinemia, fails to stimulate
protein synthesis further. While most feeding studies in humans have investigated the role of
the insulin phosphatidylinositol 3-kinase (PI3K) pathway in the activation of mTOR (73, 83,
93, 119), there is mounting evidence from cell culture studies indicating that amino acids can
activate mTOR independent of insulin via a class of regulatory GTPase proteins (280) as well
General Introduction 14
as the type III PI3K vacuole protein sorting-34 (hVps34) (146); these pathways may
represent a mechanism by which mTOR signalling molecule phosphorylation is enhanced by
dietary amino acids in humans in the absence of hyperinsulinemia (59, 100, 163).
Alternatively, the dephosphorylation of a novel and as yet unexplored in humans, site on the
guanine nucleotide exchange factor eIF2B by amino acids has been shown to occur
independently of mTOR activation in vitro (264). Finally, the role of 4E-BP1 in negatively
regulating protein synthesis may be called into question based on the recent observation that
over-expression of this protein in cultured cardiomyocytes has no effect on the rapamycin
sensitive stimulation of global protein synthesis (123). Collectively, these data demonstrate
the complexity regulation of mRNA translation and highlight the potential for multiple
pathways for the activation of muscle protein synthesis with feeding in humans.
1.3 Metabolic and cellular adaptations to endurance exercise and
nutrition For competitive endurance athletes, the desired outcome of an endurance
training regimen is to increase the ability to sustain a desired power output or speed of
movement over a given time. Therefore, training for enhancement of endurance performance
should aim to induce a myriad of physiological and metabolic adaptations that enable the
athlete to: i) increase the rate of energy production from both aerobic and oxygen-
independent pathways; ii) maintain tighter metabolic control (i.e. match ATP production with
ATP hydrolysis); iii) minimize cellular disturbances; iv) increase economy of motion; and v)
improve the resistance of the working muscles to fatigue during exercise. Key components of
a training regimen are the duration, intensity, total volume and the frequency with which
training sessions are conducted. The sum of these inputs can be termed the ‘training stimulus’
(12). When exercise training persists over an extended period of time, ‘chronic’ adaptations
occur. Although chronic adaptations in skeletal muscle are thought to result from the
General Introduction 15
cumulative effect of repeated bouts of exercise, the initial signalling responses that lead to
these chronic adaptations are likely to occur after each training session (266). It has been
established that the process of converting a mechanical signal generated during contraction to
a molecular event that promotes skeletal muscle adaptation, involves the up-regulation of
primary and secondary messengers that initiate a cascade of events that result in activation
and/or repression of intracellular signalling pathways regulating exercise-induced gene
expression and protein synthesis and breakdown (270).
The extent to which acutely altering substrate availability might modify the training
impulse has been a key research area among exercise physiologists and sport nutritionists for
a number of years. Altering substrate availability affects not only resting energy metabolism
and subsequent fuel utilization during exercise, but also the regulatory processes underlying
gene expression (7, 109). To bring about such modifications, a number of highly coordinated
processes occur, including gene transcription, RNA transport from the nucleus, protein
synthesis and, in some cases, post-translational modification of the protein.
1.3.1 Endurance exercise adaptations
Prolonged endurance training elicits a variety of metabolic and morphological
adaptations. These include; fast-to-slow fibre-type transformation (278), glycogen sparing
effects due to alterations in substrate metabolism (115), enhanced lactate kinetics and
increased mitochondrial density (125). Moreover, repeated bouts of endurance exercise alter
the expression of a host of gene products that promote adaptation toward a fatigue resistant
phenotype (3). Mitochondria are the main sub-cellular structures that determine the oxidative
capacity and fatigue resistance to prolonged contractile activity in skeletal muscle (118).
Endurance training has been shown to increase mitochondrial protein content by 50–100%
General Introduction 16
within ≈6 weeks, but a protein turnover half-life of ≈1 week means a continuous training
stimulus is required to maintain elevated mitochondrial content (278). While enhanced
oxygen kinetics, substrate transport and buffering capacity all contribute to enhanced
endurance capability in skeletal muscle, improved endurance is due primarily to increased
mitochondrial density and enzyme activity termed ‘mitochondrial biogenesis’.
1.3.2 Muscle protein metabolism after endurance exercise
Measurement of mixed muscle protein synthesis in skeletal muscle represents a
weighted average of the rates of synthesis of all proteins in the muscle, i.e. it does not
distinguish the response of different proteins constituting the mixed fraction. Intuitively,
given the divergent adaptive response to different types of training, it is safe to assume that
resistance and endurance exercise should differentially influence distinct protein fractions. At
present, relatively few studies have investigated the impact of endurance exercise on muscle
protein turnover. This may be related to the general observation that endurance exercise does
not typically result in significant gains in muscle mass. However, changes in muscle protein
synthesis following endurance exercise are relevant for tissue repair and remodelling as well
as changes in synthesis of non-contractile proteins (i.e. mitochondrial proteins) (35).
Currently, differences in exercise mode and intensity utilized in previous studies, cloud our
understanding of the protein synthetic response to endurance exercise (40, 178, 243).
It has been well documented that aerobic exercise increases whole-body (39) and mixed
muscle protein synthesis le (40, 106-108, 178, 243) (Table 1.1). Initial studies examining the
acute response of muscle proteins to aerobic exercise established that low load treadmill
exercise (at 40% V&O2max) stimulated an increase in muscle protein synthesis in untrained
General Introduction 17
subjects (40, 231). Although non-significant, Tipton et al. (243) also demonstrated that high
intensity swimming increased muscle protein synthesis by ~40% in trained female swimmers.
Studies investigating the protein synthetic response to endurance exercise have thus far
yielded variable results which may be related to the muscle studied, the mode/intensity of
exercise, or participant training status. Certainly, the latter may have considerable influence
as it has been shown chronic endurance training increases basal rates of skeletal muscle
protein turnover (208, 231). With regard to the synthesis of specific muscle protein fractions,
Miller et al. (178) reported that a unique, one-legged kicking model of endurance exercise
increased the synthesis of sarcoplasmic and myofibrillar proteins for 48 and 72 hours post-
exercise, respectively. Endurance exercise, however, is not commonly characterized by
skeletal muscle hypertrophy as would be expected with such a robust increase in myofibrillar
protein synthesis. Therefore, the “aerobic” nature of this exercise model may be more
appropriately labelled a low-intensity resistive type exercise. However, a recent study by
Wilkinson et al. (267) examined the synthetic response of myofibrillar and mitochondrial
proteins following single-leg cycling for 45 min (75% V&O2max) in both the untrained and
trained state. Following the cycle, a robust increase in mitochondrial protein synthesis
occurred regardless of training status (267). Thus, mitochondrial and to a lesser extent
sarcoplasmic proteins are the primary proteins contributing to the increase in mixed muscle
protein synthesis after endurance exercise, potentially to counteract the suppression of muscle
protein synthesis that occurs during endurance exercise (40). Whether this marked synthetic
response of mitochondrial proteins can be enhanced with post-exercise nutrient provision
remains to be elucidated.
In addition to muscle protein synthesis, muscle protein breakdown is also elevated
following acute endurance exercise (231), whilst resting rates of breakdown increase
General Introduction 18
following long-term endurance training (208). Direct measurement of muscle protein
breakdown with endurance exercise is rare and the results somewhat equivocal (74, 84, 231).
Using arteriovenous blood sampling, other investigators have demonstrated that whole-body
protein breakdown is transiently elevated after moderate intensity walking exercise,
compared with rest (231), but others report no change following exercise (243). These studies
utilized moderate to low-intensity exercise, rather than high intensity exercise. However,
studies of high-intensity endurance exercise have reported an increase in indirect markers of
muscle protein breakdown including 3-methyl-histidine excretion (40) and net amino acid
efflux from the leg (26, 253) during exercise.
1.3.3 Intracellular signalling response to endurance exercise
Expansion of skeletal muscle aerobic capacity is determined by changes in
mitochondrial protein content, observed after as little as 6-7 training sessions in humans (90).
Adaptation of mitochondrial protein content in skeletal muscle is highly complex and
involves the coordinated expression of a number molecular targets (125), the vast majority of
which are located in the nucleus (96). Thus, an important aspect of mitochondrial biogenesis
is the transport of nuclear precursor proteins into the organelle (125). Critical to the
expression of genes promoting mitochondrial biogenesis are the principles of gene regulation;
transcription initiation and interaction at the gene promoter (125). Mitochondrial biogenesis
with repeated endurance training stems from an increase in the activity of transcriptional
complexes that contain PPAR γ co-activator (PGC1α). A seminal study by Pilegaard et al.
(210) showed that acute endurance exercise increases the transcription and mRNA content of
PGC1α, an effect that is further potentiated after repeated training bouts.
Recently, control of mitochondrial oxidative function has been linked to transcriptional
control of PGC1α by mTOR (56), a critical protein implicated in the regulation of muscle
General Introduction 19
protein synthesis through translation initiation. In support of this hypothesis, others report
that endurance exercise mediates the phosphorylation of proteins implicated in mRNA
translation initiation (38, 175, 267) (i.e. mTOR) elongation (175) (i.e. eEF2). Quantitatively,
it appears that there is little difference in the extent of the acute signalling responses of leg
muscles working in different modes of exercise (38, 267). Thus, the notion that endurance
and resistance training adaptations occur through divergent molecular signalling pathways in
vitro (9, 49) may not accurately reflect the adaptive process in humans. These data suggest
that any major increase in contractile activity or possibly fuel utilization in untrained muscle
will result in the same global anabolic response.
Skeletal muscle protein breakdown is primarily mediated through the ATP dependent
ubiquitin-proteosome pathway (UPP), which degrades myofibrillar and sarcoplasmic proteins
in concert with the calpain system (13, 92). Atrogin-1 and muscle specific RING finger
protein-1 (MuRF-1) are the primary atrogenes of the UPP and are induced through FOXO
signalling (92). These factors are acutely responsive to endurance exercise, presumably as
part of the muscle remodelling process (106, 164). Although the role of these proteolytic
factors in regulating exercise-induced protein breakdown is not well defined, recent evidence
indicates the mRNA expression of MuRF-1, calpain-2, atrogin-1 and MRF4 are increased
following high-intensity resistance (164, 275) and endurance exercise (108, 164, 275).
In summary, endurance exercise elevates mitochondrial protein synthesis via enhanced
translational activity. Subsequent to this process, the activity of transcription factors and
transcriptional co-activators is critical in the regulation of mitochondrial biogenesis.
However, our current understanding of how translational and transcriptional processes act
General Introduction 20
synergistically to control mitochondrial biogenesis is limited and further studies are
warranted.
1.3.4 Muscle protein synthesis after post-endurance exercise feeding
Currently, few studies have investigated the response of muscle protein synthesis to
post-endurance exercise feeding. Levenhagen and colleagues (162) were the first to show that
the ingestion of carbohydrate plus protein immediately after a 1 hour cycle (60% V&O2max),
increased leg and whole-body protein synthesis; changes associated with a net protein gain.
However, in the study of Levenhagen et al. (162) carbohydrate-only and carbohydrate plus
protein beverages were not matched for energy intake, thus it was unclear whether the
differences observed were due to the protein per se or differences in total energy intake. To
remedy this, Howarth et al. (120) compared carbohydrate plus protein to carbohydrate-only
beverages matched for total energy and carbohydrate content. In contrast to the single bolus
feeding of Levenhagen et al. (162) beverages in this study were ingested at regular intervals
during 3 hours of post-exercise (50-80% V&O2max) recovery. The authors demonstrated that
carbohydrate plus protein increased mixed muscle protein synthesis compared with both
carbohydrate treatments, without any significant rise in insulin concentration. Despite strong
evidence that mixed muscle protein synthesis is stimulated by protein feeding rather than any
change in insulin following endurance exercise, doubt has been raised by a recent study
showing no additive effect of feeding on mixed muscle protein synthesis following a 1 hour
cycle (~72% V&O2max) (108). However, the authors (108) measured muscle protein synthesis at
2-6 hours post-exercise, 1 hour after feeding had occurred. Thus, the peak anabolic response
to exercise and feeding may have occurred earlier than was measured concealing any
potential difference (157, 268). Importantly, none of these studies (108, 120, 162) have
attempted to resolve the specific protein fractions contributing to changes in mixed muscle
General Introduction 21
protein synthesis. Furthermore, it is possible that the measure of mixed muscle protein
synthesis may not be sensitive enough to reflect changes in the synthesis of specific proteins
(108). Recently, Moore et al. (181) demonstrated that the synergism of protein ingestion and
resistance exercise augmented myofibrillar protein synthesis. Thus, it seems that protein
nutrition is more than merely a substrate, but instead is an input into a system that affects
phenotype due to the influence it exerts in regulating muscle protein synthesis. These data
have led Howarth et al. (120) to suggest that the increase in mixed muscle protein synthesis
with protein ingestion following endurance exercise could be due, in large part, to an increase
in the synthesis of mitochondrial proteins. To date, this thesis has yet to be confirmed. A
summary of studies investigating the effect of a single bout of endurance exercise on protein
metabolism are presented in Table 1.1.
1.3.5 Intracellular signalling response to post-endurance exercise feeding
Despite the permissive role of insulin on muscle protein synthesis, it has been
suggested that elevated plasma insulin levels are required to fully activate the translation of
mRNA, initiated following endurance exercise (158). The suggestion is that insulin activates
mTOR, via an upstream pathway involving PI3K and Akt (72, 147). Interestingly, relatively
new findings from cell culture and drosophila experiments demonstrate that essential amino
acids can directly stimulate mTOR via hVps34 (36) as well as through the Ste20 protein
kinase, MAP4K3 (78). A family of small GTPases known as Rag proteins may also be
important in promoting the intracellular localization of mTOR towards Rheb during amino
acid stimulation, though this has only been observed in human embryonic kidney cell cultures
to date (221). In summary, these data suggest that insulin and amino acids act independently
to regulate mTOR. However, following carbohydrate plus protein feeding, amino acid-
General Introduction 22
induced mTOR activation appears to be crucial for increasing protein synthesis as opposed to
the rise in insulin concentration (5, 6).
Relatively few studies have investigated the response of intracellular signalling to
carbohydrate plus protein feeding following endurance exercise. Ivy et al. (126) compared the
intracellular signalling response to carbohydrate plus protein feeding ingested over 45
minutes after a 1 hour variable intensity cycle. The authors demonstrated that carbohydrate
plus protein activated mTOR and rpS6 compared with a non-energetic placebo. However, the
study design of Ivy et al. (126) did not investigate the response of protein or carbohydrate
feeding alone. Thus the authors could not say with any certainty whether the effects of the
drink on intracellular signalling were due to the greater amino acid availability, elevated
plasma insulin, or a combination of the two. The same research group, (186) had earlier
conducted a more rigorous experiment in which they showed that ingesting carbohydrate or
protein after exhaustive exercise transiently increased the phosphorylation of mTOR, 4E-
BP1, rpS6 and p70S6K in rats. However, the phosphorylation of rpS6 and 4E-BP1 was
sustained when carbohydrate and protein were co-ingested. Taken together, these data
demonstrate that feeding either carbohydrate or protein alone following intense endurance
exercise increases the phosphorylation of proteins implicated in translation initiation, but not
to the extent of carbohydrate and protein provided together. On the other hand, recent
evidence indicates that increasing levels of circulating insulin in the post-endurance exercise
period through feeding interventions, may suppress the phosphorylation of proxy markers of
muscle proteolysis (108). However, in the absence of muscle protein turnover data to support
the intracellular signalling response, it difficult to make definitive conclusions regarding the
precise role of post-endurance exercise nutrient ingestion on muscle protein breakdown.
Study
Endurance
protocol
Exercise
duration
Subject training
status
Protein
synthesis
Protein
breakdown
Intracellular
signalling
Carraro et al. 1990
(40)
40% VO2max
4 hours
Healthy young males
• 96% ↑ in mixed MPS **
• 85% ↑ in 3MH post-ex
*
N/A
Tipton et al. 1996
(243)
~85-90% HRmax ~1.5 hours Trained female
swimmers • 41% ↑ in mixed MPS
• ↔ in WBPB at 3 h
post-ex
N/A
Sheffield-Moore et al.
2004 (231)
40% VO2max 45 min Untrained elderly and
young males • ~ 65-75% ↑ in mixed
MPS *
• ↑ in WBPB at 10 min
post-ex *
N/A
Miller et al. 2005
(178)
~67% Wmax
1 hour Healthy young males • 52% ↑ in SARC MPS *
• 70% ↑ in MYO MPS *
N/A N/A
Mascher et al. 2007
(175)
75% VO2max 1 hour
Healthy young males N/A N/A • ↑ Akt, mTOR,
p70S6K, AMPK,
• ↓ eEF2 *
Ivy et al. 2008 (126)
40-90% VO2max 1 hour Healthy young males N/A N/A • ↑ rpS6, p70S6K *
Wilkinson et al. 2008
(267)
75% VO2max 45 min
Healthy young males • ↑ Mitochondrial MPS * N/A • ↑ mTOR, p70S6K,
FAK, eIF4E, AMPK
*
Harber et al. 2010
(108)
~72% VO2max 1 hour Healthy young males • 57% ↑ in mixed MPS *
N/A • ↑ MuRF-1, MRF4,
calpain-2 *
Harber et al. 2010
(107)
75% VO2max 45 min Trained males • ~37% in mixed MPS *
N/A N/A
Table 1.1 Comparison of studies examining muscle protein metabolism and intracellular signalling following endurance exercise
V&O2max; maximal oxygen uptake, HRmax; maximum heart rate, Wmax; maximal work load, MPS; muscle protein synthesis, SARC; sarcoplasmic,
MYO; myofibrillar, WBPB; whole-body protein breakdown, 3MH; 3-methyl histidine, MuRF-1; muscle specific RING finger-1, MRF4; muscle
regulatory factor 4, post-ex: post-exercise, ↑; increase, ↓; decrease, ↔; no change, *; P < 0.05, **; P < 0.01.
General Introduction 24
1.4 Influence of protein nutrition on endurance performance and recovery
Nutritional strategies that improve performance and assist recovery are unique to each
sporting event and individual. Although most athletes can satisfy their nutritional
requirements before and/or after exercise, long-duration activities require that participants
also address their nutritional needs during exercise. Endurance exercise promotes vast
increases in energy utilization, with significant increases in carbohydrate and fat oxidation
rates (32, 53). Sizeable losses of fluid and electrolytes from sweat may also occur,
particularly during prolonged exercise in the heat (192). As a result, inadequate fluid and
nutrient intake during endurance exercise can lead to dehydration, hyponatremia, glycogen
depletion, hypoglycemia and impaired performance. In addition, nutritional deficiencies
during prolonged activity may limit the capacity for rapid recovery after exercise, which may
affect subsequent performance. Numerous studies have investigated nutritional approaches to
minimize these issues, resulting in the emergence of strategies that elicit positive effects for
endurance athletes. For endurance athletes, carbohydrate feeding is the most commonly used
nutritional strategy. It is generally agreed that carbohydrate beverages are effective in
promoting fluid balance and euglycemia and augmenting performance during prolonged
endurance activities (50, 52, 133, 248). Typical guidelines suggest ingesting sports beverages
with 4 - 8% carbohydrate at regular intervals during exercise to provide approximately 600 -
1400 mL of fluid and 30 - 60 g of carbohydrate per hour (4, 50, 52). The traditional role of
carbohydrate sports beverages has been to optimize performance by delaying dehydration and
hypoglycemia and potentially influencing glycogen depletion and central fatigue. However,
nutrient intake during prolonged exercise may also have important implications for recovery
from exercise.
General Introduction 25
Recent interest has centred on the use of protein nutrition in sports beverages for
endurance athletes. Specifically, it has been suggested that the co-ingestion of carbohydrate
with protein during exercise, improves endurance performance. Moreover, recent studies
indicate that carbohydrate-protein co-ingestion during and following exercise may reduce
indices of muscle damage, thus enhancing the recovery of subsequent performance and
muscle function.
1.4.1 Carbohydrate-protein co-ingestion for endurance exercise performance
Several studies have shown that carbohydrate plus protein ingestion can extend
endurance time-to-exhaustion in the range of 13-36% (127, 222, 223). More recently,
Saunders et al. (224) suggested the addition of protein to carbohydrate specifically improved
late-exercise time-trial performance. However, a number of methodological differences
between studies make it difficult to discern whether any benefits observed for carbohydrate
plus protein are the result of a protein-mediated effect.
First, there are discrepancies in the way exercise performance has been assessed in
previous studies. The ecological validity of exhaustive exercise protocols (used in (127, 222,
223)) is limited, as endurance athletes do not compete in events that require sustaining a fixed
power output for as long as possible. Further, exhaustive exercise tests display a large
variation between repeated bouts of ~26% (132), which may be exacerbated in lesser trained
participants (57). Moreover, claims that the addition of protein improves late-exercise
performance were based on a time-trial protocol that was not specifically designed to record
power output and measure late-stage exercise performance (224). On the contrary, time-trial
General Introduction 26
cycle tests have been found to be highly reproducible and sensitive to small changes in
exercise performance (132, 160, 201). Secondly, additional energy consumed when protein is
added to a carbohydrate containing beverage may explain the performance benefits observed
by others. Indeed, studies in which endurance capacity was extended with additional protein,
fed a carbohydrate dose that may have been too low (<50 g·hˉ1) to attain peak exogenous
oxidation rates (134). Thus, if carbohydrate is consumed at a rate considered optimal for
exogenous carbohydrate oxidation, the role of protein for improving endurance performance
may be negligible (252). Finally, the control of external variables has varied greatly between
previous studies. Knowledge of parameters including; i) time elapsed, ii) distance travelled
and iii) heart-rate during exercise may compromise the blinding of treatments, creating a
placebo effect. Early studies did not report the control of these conditions (127, 219, 222-224,
251). In contrast, investigations that implement a strictly controlled exercise environment
reported no effect of protein co-ingestion on time-trial performance (199, 252).
In addition to methodological considerations, there is currently no plausible mechanism
to explain the reported performance benefits when protein is added to a carbohydrate
beverage. It has been suggested that the addition of protein to carbohydrate may enhance
endurance performance through one or a combination of several putative mechanisms. These
include; i) altering substrate utilization in a glycogen depleted state (i.e. during late-exercise)
(224); ii) offsetting the decline in TCA cycle intermediates during prolonged exercise iii)
delaying the onset of central fatigue and iv) facilitating fuel transport via an increase in fluid
absorption. Currently, the efficacy of adding protein to carbohydrate for endurance
performance remains unclear.
General Introduction 27
1.4.2 Carbohydrate-protein co-ingestion for indirect markers of muscle damage and
recovery
The traditional role of carbohydrate feeding in close proximity to exercise has been to
optimize performance by delaying dehydration and hypoglycemia as well as potentially
influencing glycogen depletion and central fatigue. However, nutrient intake during
prolonged exercise is thought to be effective in ameliorating indices of muscle damage,
thought to impair the rate of recovery. Thus, a number of studies have reported that
carbohydrate plus protein co-ingestion during and following endurance exercise improves
several indices of recovery. Specifically, these studies demonstrate that the addition of
protein to carbohydrate prolongs subsequent time-to-exhaustion in the range of 40-55% (222,
233, 269). Others have found additional protein results in small increases in the recovery of
muscle function, on the order of 1-2 knee extension lifts (251) and 1-2cm in vertical jump
height (251). In contrast to these data, several studies failed to find improvements in recovery
of subsequent performance, assessed using various methods (19, 98, 165, 177, 219). Thus, the
impact of adding protein to carbohydrate on recovery from intense exercise is considered to
be somewhat equivocal.
Reductions in the concentration of proxy markers of muscle damage such as plasma
creatine kinase (91, 165, 177, 219, 222, 223, 233, 251) and lactate dehydrogenase (165, 219)
are thought to play a key role in aiding the recovery process when protein is added to
carbohydrate. Moreover, several studies suggest that the addition of protein to a carbohydrate
beverage may improve recovery through attenuating subjective ratings of muscle soreness
(165, 177, 219, 233). However, evidence to support an effect of additional protein for
improving recovery is inconsistent with several investigations finding no difference in the
post-exercise rise in plasma creatine kinase (20, 98), muscle soreness (20, 177) and the
General Introduction 28
recovery of endurance capacity (19) when additional protein is ingested. Thus, although
many endurance athletes now considered it a ‘necessity’ to consume protein following
exercise, the efficacy of protein supplementation as a tool to improve recovery is unclear, due
in large part, to the applicability of different recovery assessments to the athletic environment
and a limited understanding of the mechanisms facilitating these purported improvements. A
comparison of studies investigating the impact of carbohydrate-protein co-ingestion on
endurance performance and indices of recovery is highlighted in Table 1.2.
Study
Endurance
protocol
Beverages
Timing
CHO
(g·h-1
)
PRO
(g·h-1
)
Fluid
(mL·h-1
)
Performance
outcome
Recovery
outcome
Ivy et al. 2003 (127)
• TTE at 85%
VO2max.
CHO
C+P
During Ex
45
45
-
4
600
• 36% ↑ in TTE for C+P *
N/A
Saunders et al. 2004
(222) • TTE at 75% VO2max
• TTE at 85% VO2max
12-15 h later
CHO
C+P
During and
immediately
post-Ex
37*
37*
-
9*
500* • 29% ↑ in 1st TTE for C+P
*
• 40% ↑ in 2nd
TTE for C+P
*
• 83% ↓ in plasma CK for
C+P *
Romano-Ely et al.
2006 (219) • TTE at 70% VO2max
• TTE at 80% VO2max
24 h later
CHO
C+P
During and
immediately
post-Ex
56 *
45 *
-
11*
560*
None • 65% ↓ in CK for C+P *
• 66% ↑ in MS for CHO *
Luden et al. 2007
(165) • 5km race after 6
days of
supplementation
•
CHO
C+P
Immediately
post-training
102*
102*
-
26*
1400*
None • ↓ MS and CK for C+P *
van-Essen &
Gibala.2006 (252)
• 20km cycle time-
trial
CHO
C+P
During Ex 60
60
-
20
1000 None N/A
Osterberg et al. 2008
(199) • Pre-loaded cycle
time-trial
CHO
C+P
During Ex 60
60
-
20
1000 None N/A
Valentine et al. 2008
(251) • TTE at 75% VO2max CHO
CHO
C+P
PLA
During Ex 78
100
78
-
-
-
12
-
1000 • C+P and CHO ↑ TTE *
• CHO TTE similar to PLA
• ~20% ↑in LE for C+P *
• ~50% ↓ in CK for C+P
*
Saunders et al. 2007
(223)
• TTE at 75% VO2max CHO
C+P
During and
immediately
post-Ex
41*
41*
-
10*
560*
• 13% ↑ TTE for C+P *
• ~45% ↑ in CK for CHO
*
Table 1.2 Comparison of studies examining endurance performance and recovery following carbohydrate-protein co-ingestion
TTE; time-to-exhaustion test, V&O2max; maximal oxygen uptake, Ex; exercise, CHO; carbohydrate, PRO; protein, C+P; carbohydrate plus protein, PLA;
placebo, *; average values based on 70kg adult, CK; plasma creatine kinase, MS: muscle soreness, LE; leg extension strength, *; P < 0.05.
General Introduction 30
1.5 Glucose metabolism in skeletal muscle
Insulin stimulates the uptake of glucose from the circulation into many body tissues, of
which, skeletal muscle accounts for ~75-80% of whole body insulin-stimulated glucose
uptake (63, 232). Thus, skeletal muscle is considered as the primary tissue for regulating
glucose homeostasis. At the cellular level, insulin stimulates glucose uptake into skeletal
muscle by promoting glucose transporter protein (GLUT4) translocation to the cell
membrane through the activation of an insulin-dependent signalling pathway.
1.5.1 Intracellular regulation of glycemic control in skeletal muscle
Appropriate signalling through the insulin pathway is critical for the regulation of
plasma glucose levels in skeletal muscle. The binding of insulin to its receptor leads to the
tyrosine phosphorylation of the insulin receptor (IR), the insulin receptor substrate-1 (IRS-1)
and hence, to the activation of PI3K. The activation of PI3K leads to the production of
phosphatidylinositol-(3, 4, 5)-P3 (PIP3), a key secondary messenger, which recruits 3-
phosphoinositide-dependent protein kinase (PDK) to the plasma membrane (257). PI3K
activity also induces the activation of downstream signalling intermediates; Akt and atypical
protein kinase C (PKC) (257). In skeletal muscle, Akt mediates the insulin-stimulated
inactivation of glycogen synthase kinase-3 (GSK3) by phosphorylation, thereby allowing
glycogen synthase (GS) to become dephosphorylated and activated (55). Activation of PI3K
is known to result in ribosomal protein S6 kinase (p70S6K) activation, a mechanism
involving mTOR (212), which is inhibited by the blocking agent rapamycin. p70S6K can also
be directly phosphorylated by PDK in a rapamycin-independent manner (11) and may play an
important role in mediating GSK3, thus promoting the activation of glycogen synthase. The
role of Akt in the regulation of insulin stimulated glucose transport is strengthened by the
General Introduction 31
recent discovery of AS160, a 160-kDa protein which is phosphorylated by Akt (140). AS160
contains a GTPase activating domain for Rabs, which are small G proteins required for
membrane trafficking (including GLUT4 translocation). It has been shown that pAS160 in rat
skeletal muscle is rapidly increased when exposed to a physiological increase in insulin (34).
Furthermore, insulin-stimulated phosphorylation of AS160 occurs in a PI3K-Akt-dependent
manner (65, 141).
1.5.2 Type II diabetes and insulin resistance
The prevalence of diabetes mellitus has reached global epidemic proportions. It is
predicted that by 2025, 300 million people will have diabetes (279). The majority of diabetic
patients (over 90%) suffer from Type II diabetes, a progressive metabolic disorder with a
slow and insidious onset. The incidence of Type II diabetes and associated metabolic
conditions stems from societies adopting ever more sedentary lifestyles in the face of an
excessive energy intake. Consequently, diabetes imparts major health consequences at both
individual and public health levels (136). Although several pharmacologic approaches to the
treatment of Type II diabetes are available, there is growing interest in developing non-
pharmacological interventions to improve glycemic control. Lifestyle interventions such as
exercise and nutrition that delay, or even prevent, Type II diabetes have the potential to
improve the health of the population and reduce the economic burden on healthcare services.
The pathophysiology of Type II diabetes develops primarily from i) defects in skeletal
muscle sensitivity to insulin ii) defects in liver sensitivity to insulin and iii) pancreatic β cell
dysfunction. Collectively, these defects are referred to as the Triumvirate (64). Under normal
General Introduction 32
circumstances, insulin binds to insulin receptors on target organ cells, resulting in a series of
cellular events that promote intracellular glucose transport and metabolism (206). Insulin
resistance describes the diminished sensitivity of cells to the signalling effects of insulin. It is
generally accepted that resistance to insulin in target tissues, primarily skeletal muscle,
precedes the development of Type II diabetes (204). In insulin resistant states, the pancreas
compensates by secreting increased amounts of insulin to maintain normoglycemia. This
resistance is followed by a decrease in insulin secretion as a result of progressive pancreatic
β-cell dysfunction, leading to the onset of overt diabetes with fasting hyperglycemia.
Comprehensive discussion of the mechanisms underpinning insulin resistance in liver tissue
and β cell dysfunction is beyond the scope of this thesis chapter. Instead, the interested reader
is referred to the following literature (62, 64). Thus, in the current chapter, the mechanisms
responsible for insulin resistance will be discussed within the context of skeletal muscle
tissue.
1.5.3 Mechanisms of insulin resistance in peripheral tissues
The intracellular signalling defects underlying insulin resistance in skeletal muscle are
now becoming apparent. Signalling defects at the level of the insulin receptor are unlikely to
fully account for the impairments in glucose transport in skeletal muscle from Type II
diabetic patients (46, 154). Downstream of the insulin receptor, impaired insulin-stimulated
PI3K activity in skeletal muscle has been confirmed in studies of obese (154, 249) and Type
II diabetic patients (58, 144, 145). Defects in insulin signalling at the level of IRS-1 and PI3K
probably account for the impaired insulin action on PKC reported by others (17, 145).
Current investigations regarding cellular defects at the level of Akt in insulin resistant states
have yielded conflicting results (17, 145, 155). These discrepant findings are likely due to
General Introduction 33
fundamental differences in cell culture and human study models. Thus, a combination of
relatively minor defects downstream of IRS phosphorylation may act synergistically to cause
insulin resistance and impair glucose tolerance, defined as the ability of skeletal muscle and
the liver to remove glucose from the circulation (137).
Along with skeletal muscle, adipose tissue is also an important site for insulin-
stimulated glucose uptake (47). In addition to Type II diabetes, insulin resistance is also
commonly associated with obesity, hypertension and cardiovascular disease. Many studies
have revealed an association between increased lipid availability and insulin resistance,
suggesting that there is a causative link between the two. Studies demonstrate that high-fat
feeding is closely associated with the accumulation of intramuscular triacylglycerol (IMTG)
(68, 143, 198, 237). Moreover, insulin resistance can occur through short term
(physiologically relevant) increases in lipid availability. However, it appears unlikely that the
IMTG accumulation per se directly impairs insulin action (33). This suggestion is further
supported by the observation that endurance trained (highly insulin sensitive) individuals
have elevated IMTG content. Instead, it is believed that intermediates in fatty acid
metabolism, such as fatty acyl-CoA (198), ceramides (104) or diacylglycerol (DAG) (241)
and an elevated inflammatory state, link obesity to compromised insulin signalling in the
muscle. DAG is identified as a potential mediator of lipid-induced insulin resistance (113,
214). Increased DAG levels are associated with PKC activation and a reduction in both
insulin-stimulated IRS-1 tyrosine phosphorylation and PI3K activity (276). Recently,
Newgard et al. (187) demonstrated the accumulation of a number of acylcarnitines in skeletal
muscle of high-fat fed rats supplemented with additional branched chain amino acids
(BCAA’s) compared with non-supplemented rats. Thus, in the context of high-fat
consumption, supplementation of protein and/or amino acid may contribute to obesity-
General Introduction 34
induced insulin resistance. The efficacy of protein ingestion on FFA metabolism during a
period of reduced energy intake provides an interesting avenue for future studies.
1.6 Influence of exercise on glucose homeostasis
Insulin resistance and Type II diabetes are strongly associated with a decline in physical
inactivity (70, 153). However, despite the underlying metabolic and molecular defects the
precipitate insulin resistance, skeletal muscle contraction can effectively restore glycemic
control in a manner independent of insulin (111). Moreover, these properties are preserved in
individuals with Type II diabetes (97, 142). Thus, at best, exercise represents an effective
means of preventing insulin resistance and Type II diabetes, but perhaps more realistically,
represents a means by which to attenuate the metabolic symptoms associated with these
conditions. Long-term intervention studies (3-5 years) show that Type II diabetes ‘risk
reduction’ is further reduced when physical activity is implemented in combination with
dietary modifications (75, 202). More importantly, increased physical activity and reduced
energy intake are associated with a greater reduction in the incidence of diabetes compared
with pharmacological interventions such as metformin (a suppressor of endogenous glucose
production) (250).
1.6.1 Resistance exercise and glycemic control
Physical inactivity induces a wide variety of changes in both whole-body functional
capacity and muscle metabolism (220), with most of the metabolic effects of physical
inactivity on insulin resistance being rapid in onset and of relatively short duration (97).
Studies investigating the role of physical activity as a means to improve the insulin sensitivity
have generally applied endurance exercise as a model. Indeed, a single bout of moderate-to-
General Introduction 35
high intensity endurance exercise has been shown to acutely improve insulin sensitivity
and/or glucose tolerance (66, 67, 176, 205). This effect has been reported to persist for a
period ranging from 2 hours (176), 4–6 hours (273), 12–16 hours (66, 67) to 48 hours post-
exercise (176, 205). In contrast to endurance exercise, limited information is available
regarding the impact of resistance exercise on glucose metabolism. It has been demonstrated
that 6-12 weeks of resistance training lowers the percentage of glycosylated haemoglobin and
increases glucose disposal in Type II diabetics due, in large part, to muscle hypertrophy and
an increase in the protein content of insulin-signalling intermediates (54, 77, 117). In
contrast, the effectiveness of a single bout of resistance exercise on glycemic control and the
mechanisms regulating this response are less well understood. Whereas some authors have
reported an improved glucoregulatory response 12–24 hours after a single bout of resistance
exercise (77, 81, 150, 258), others have failed to observe any change in insulin sensitivity
(44, 166). These discrepant findings may be related to the method utilized to determine
glucose and insulin kinetics (i.e. oral glucose tolerance tests vs. insulin tolerance test) or
differences in the exercise stimulus and participant training status. Nevertheless, these data
strongly suggest that a single bout of resistance exercise of sufficient volume and intensity
can improve insulin sensitivity and glycemic control in humans.
1.6.2 Intracellular signalling regulating glucose uptake with acute exercise
The molecular mechanisms for enhanced glucose uptake and insulin sensitivity with
exercise are related to the increased expression and/or activity of key enzymes and signalling
proteins involved in skeletal muscle glucose and fat metabolism (69, 277). Prolonged
improvements in insulin sensitivity and glucose transport may reflect changes in protein
expression (i.e. enhanced or suppressed) that occur following each acute exercise bout. The
immediate effects of resistance exercise on skeletal muscle glucose transport have long been
General Introduction 36
thought to occur via AMPK regulation of GLUT4 trafficking to the cell membrane (71, 240)
rather than through any enhancement of proximal insulin signalling steps (122, 273).
However, it has recently been demonstrated that contractile activity can promote GLUT4
translocation irrespective of AMPKα2 (159). In this regard, recent work has implicated
sucrose non-fermenting AMPK-related kinase (SNARK) protein as an novel and potentially
important mediator of skeletal muscle glucose uptake (149). Currently, the precise signalling
events that promote glucose uptake in skeletal muscle are unclear.
The recent discovery of the most distal signalling proteins implicated in insulin-
stimulated GLUT4 transport, AS160 and TBC1D1 (from the same family domain of
proteins), has provided us with a clue as to how contraction-induced AMPK activity regulates
GLUT4 translocation. Phosphorylation of AS160/TBC1D1 is thought to lead to inhibition of
AS160’s activation of Rab-GTPase proteins associated with the GLUT4 vesicle and/or cause
AS160 to disassociate from the GLUT4 vesicle. This mechanism of action thereby allows for
the accumulation of Rab GTP, thus favouring GLUT4 translocation (42, 140). Several
studies, using specific antibodies for individual phospho-sites on AS160, have reported an
increase in AS160 phosphorylation following resistance exercise (71, 121, 246). Furthermore,
when isolated skeletal muscle is treated with AICAR (an AMPK activator), or subjected to
contraction in situ, both AS160 and TBC1D1 phosphorylation are increased (34, 152, 239).
Conversely, when AMPK is inhibited (via knockout of AMPKα2), contraction-stimulated
AS160 phosphorylation is significantly reduced (152). Recently it has been hypothesized that
a large proportion of the contraction-stimulated increase in glucose transport may be
attributed to TBC1D1, whereas AS160 may play a more prominent role in regulating insulin-
stimulated glucose uptake (42). To date, this thesis has yet to be confirmed in humans. At
present, methodological limitations regarding non-specific antibody detection of
General Introduction 37
AS160/TBC1D1 cloud our understanding of the phospho-specific response of these two
proteins to insulin and contraction stimuli (42). Indeed, it has recently been demonstrated in
mice that contraction-stimulated skeletal muscle glucose uptake is regulated by site-specific
phosphorylation of TBC1D1 (259).
In summary, it has been hypothesized that AS160 and TBC1D1 act as a nexus for
insulin-dependent and insulin-independent (i.e. contraction) signalling pathways on GLUT4
translocation and skeletal muscle glucose uptake (43). Consequently, the intracellular signals
emanating from contracting muscle during exercise may interact with the insulin signalling
pathway at the level of AS160/TBC1D1, thereby acting synergistically to enhance glucose
uptake (43). In support of this suggestion, Treebak et al. (245) recently confirmed that AS160
phosphorylation was increased by prior exercise, both before and immediately after insulin
stimulation.
1.7 Influence of protein nutrition on glucose homeostasis
Concepts regarding dietary changes that prevent or assist in the management of Type II
diabetes are beginning to emerge. In the last decade the role of protein feeding as an
intervention to reduce hyperglycemia has received considerable attention. Previously, when
dietary recommendations for the general population, or for persons with diabetes were
considered, the major focus was the relative amounts and types of carbohydrate and fat (1).
Protein nutrition is generally considered in the context of that necessary to maintain, or gain
lean body mass, i.e., that needed to promote a positive net muscle protein balance. However,
in the early 1900’s, it was reported that the ingestion of egg white protein in a single-meal
study did not elevate blood glucose concentration in healthy humans (130). In persons with
General Introduction 38
Type II diabetes, it has been suggested that protein ingestion lowers postprandial glucose
concentrations (193, 196). Long-term, protein-rich diets are associated with better glycemic
control and plasma lipid profile in obese and insulin resistant individuals and promote energy
expenditure and greater relative fat reduction, compared with energy matched, high
carbohydrate or high fat diets (15, 161, 265). Thus, it appears that protein nutrition could play
an important role in glycemic control in humans.
1.7.1 Insulin-stimulated hypoglycemia with protein co-ingestion
Several decades ago Floyd and Fajans (76, 79, 80) showed that the infusion of high
doses of amino acids potentiated the release of insulin. In contrast, oral ingestion of protein or
amino acids, in an amount likely to be ingested in a high protein meal, does not alter the
insulin response (87, 88, 138, 139, 194, 197). Alternatively, co-ingesting protein with
carbohydrate does stimulate a greater insulin release than the ingestion of either
macronutrient alone. The synergistic effect of protein plus carbohydrate on insulin release
was first described in the 1960’s (200, 215) and later confirmed by Nuttall and colleagues
(195, 196). Studies conducted in vitro (24, 25, 168, 169, 227, 228, 230) and in humans (76,
226) demonstrate that amino acids co-ingested with carbohydrate potentiate insulin release
from pancreas β cells. Increasing postprandial endogenous insulin release may lower blood
glucose levels by assisting the process of skeletal muscle glucose uptake via the insulin-
dependent signalling cascade that, ultimately, enhances glucose transporter protein
translocation to the cell membrane. Thus, protein co-ingestion may alleviate many side
effects of diabetes (e.g. cardiovascular disease risk, glycation of tissues/neurones,
inflammation) and postponing an individuals’ dependency on exogenous insulin therapy.
General Introduction 39
Several notable studies from the Dutch group of Luc Van-Loon have focussed on the
insulinotropic effect of protein plus carbohydrate ingestion in Type II diabetics. Initially, this
group demonstrated that the insulin response to carbohydrate intake can be nearly tripled by
the addition of a free amino acid/protein mixture (254). Subsequently, the group showed that
this free amino acid/protein plus glucose mix augmented insulin release and lowered plasma
glucose excursions in Type II diabetics (173). With the use of a labelled glucose tracer
infusion during drink ingestion, the authors attributed the reduction in plasma glucose to an
increase in the rate of glucose disposal from the circulation, presumably taken up by skeletal
muscle. However, recent work from this group showed that co-ingesting protein with each
main meal (three times a day) did not reduce the prevalence of hyperglycemia over the course
of the day in Type II diabetics (172). To explain this finding, the authors posit that the
increase in total energy intake (~14%) when additional protein was co-ingested may have
offset any hypoglycemic effect. Thus, protein ingestion may be best implemented in the diet
in place of, rather than in addition to, other macronutrients. Nevertheless, these data provide
overwhelming evidence that co-ingesting protein with carbohydrate promotes
hyperinsulinemia. Thus, whilst the sensitivity of the pancreas to glucose is significantly
impaired in persons with long-standing Type II diabetes (211), the capacity to secrete insulin
when protein is co-ingested with carbohydrate remains functional (171, 173). However, the
efficacy of protein co-ingestion as a non-pharmacological treatment for Type II diabetes in
daily, free-living conditions is less than clear, although Manders et al. (172) hypothesized that
the presence of free amino acids, leucine in particular, may be important in stimulating
hyperinsulinemia (170).
1.7.2 Protein absorption and hydrolysis for glucose homeostasis
General Introduction 40
The co-ingestion of most common food sources of protein with glucose, promotes
hypoglycemia in Type II diabetics (86). However, the insulinotropic response is dependent on
the protein source consumed (148, 193, 195). Differences in insulinotropic and glycemic
properties of various protein sources are likely to be associated with; i) the post-absorptive
pattern of amino acid appearance in the circulation and the degree of hydrolysis of the protein
source.
Casein proteins are digested slowly and induce a lower but more prolonged
hyperaminoacidemia, whereas whey protein, which is digested quickly, induces higher
postprandial retention of amino acids (29, 190). The greater insulinotropic response to whey
protein feeding may be connected to more rapid digestion kinetics. The suggestion that whey-
induced hyperaminoacidemia is important in glucose homeostasis is supported by studies
showing that glycogen content in liver (184) and skeletal muscle tissue (185) of whey protein
fed rats, was greater than rats fed casein. In addition to the absorption kinetics, the degree of
hydrolysis of ingested proteins may be an important determinant for maintaining
normoglycemia. It is generally accepted that di- and tri-peptides, remaining after the initial
peptidase digestion, are absorbed intact (101). Indeed, whey, egg and casein hydrolysates,
containing mostly di- and tri-peptides, are more rapidly absorbed than their respective intact
protein sources (102). However, there was no difference in the glucose and insulin response
between animal and vegetable protein hydrolysates co-ingested with glucose (37, 48).
Collectively, these data imply that the rate of amino acid appearance in the circulation,
dependent on the protein source and state of hydrolysis, is an important determinant of the
insulinotropic and hypoglycaemic response.
1.7.3 Insulinotropic properties of free amino acids
General Introduction 41
In addition to protein co-ingestion with carbohydrate as a means to promote
hyperinsulinemia, it is now clear that certain free amino acids act as direct insulin
secretagogues (76, 225, 226). Nilsson et al. (190) first reported a correlation between the
postprandial insulin and early increments in leucine, valine, lysine, and isoleucine after the
consumption of different protein containing food sources. These data are further supported by
studies demonstrating that the insulinemic response to BCAA’s plus glucose co-ingestion
mimics the glycemic and insulinemic response to ingesting intact whey protein with glucose
(183, 189). Based on these data, it is apparent that the BCAA’s are the most potent insulin
secretagogues (45, 124, 255). Mechanistically, in vivo and in vitro work indicates that the
BCAA leucine both stimulates and enhances pancreatic β cell insulin secretion through
oxidative decarboxylation and allosterically activating glutamate dehydrogenase (188, 229).
The precise mechanisms, by which amino acids promote insulin exocytosis from the
pancreas, although not yet fully elucidated, likely involve depolarization of the plasma
membrane via energy sensing Ca+2
and k+ channels (188, 256). At present, the impact of
BCAA on glucoregulation in healthy and Type II diabetics is still the subject of some debate.
Manders and co-workers (170) fed a carbohydrate plus protein beverage with or without
additional leucine to Type II diabetics and reported no difference in the response of insulin or
plasma glucose.
1.7.4 Mechanisms of glucose uptake with amino acids
The signalling events that facilitate the glucose uptake in skeletal muscle are highly
sensitive to a number of stimuli, including amino acids, and can be ‘turned on’ in the
presence and absence of insulin. Surprisingly little is known about the mechanisms regulating
glucose homeostasis following protein and amino acid ingestion in healthy and Type II
diabetic humans. In contrast, the last decade has seen the emergence of rat and cell culture
General Introduction 42
studies in which glucose uptake and the associated regulatory signalling mechanisms have
been determined following treatment with different proteins and free amino acids, ingested
alone or with glucose. The main findings of these studies are summarized in Table 1.3.
Amino acids, particularly leucine, enhance the phosphorylation of mTOR and can act
synergistically with insulin to fully activate mTOR (5, 105, 203). However, insulin and
amino acids signal to mTOR via divergent pathways (105). Initially, Armstrong et al. (8)
reported that amino acids promote glycogen synthesis and glucose uptake in cultured human
muscle cells via non-insulin dependent pathways via transient inhibition of GSK3 through
phosphorylation of p70S6K. Furthermore, amino acid-stimulated glycogen synthesis was
blocked by the mTOR inhibitor rapamycin (8). Others (207) also demonstrate that the
provision of leucine stimulates glycogen synthesis in L6 myotubes in an mTOR dependent
manner. The importance of the mTOR-p70S6K pathway and the amino acid leucine for
glucose uptake is strengthened by Nishitani et al. (191). In rats with cirrhosis, a condition
characterized by impaired glucose metabolism, these authors demonstrated that leucine and
isoleucine enhanced glucose uptake, thus suppressing plasma glucose excursions compared
with saline fed controls. In soleus muscles from these rats, leucine and isoleucine were shown
to increase GLUT4 translocation to the cell membrane in insulin-free conditions. Taken
together, these studies demonstrate an important regulatory role of the mTOR-p70S6K
pathway in amino acid-stimulated glucose uptake. Intracellular regulation of glycemic control
via insulin and BCAA is summarized in Figure 1.2.
Insulin is a potent activator of muscle glycogen synthesis owing to its stimulatory effect
on glucose transport. When BCAA are provided in the presence of an elevated insulin
response, glucose uptake is potentiated through an increase in the phosphorylation of insulin-
dependent signalling intermediates Akt, PI3K and PKC (182, 184). With regard to mTOR
General Introduction 43
Figure 1.2 A simplified schematic representation of insulin-, BCAA- and contraction-
stimulated signalling mechanisms regulating glucose uptake in skeletal muscle. Dashed arrows
indicate BCAA-stimulated signalling activation. Solid arrows indicate insulin- and
contraction-stimulated signalling activation. Proteins have been labelled to designate them as
positive contractile (blue), positive feeding-induced (green) or negative (red) regulators of
glucose uptake.
signalling, evidence suggests that insulin and amino acids act synergistically to fully activate
this protein (105, 203) and that both stimuli signal to mTOR via divergent pathways (105).
Whereas amino acid feeding in insulin-free conditions appears to promote glycogen synthesis
via mTOR signalling, in the presence of insulin, BCAA-stimulated mTOR phosphorylation
has been shown to be rate-limiting for skeletal muscle glucose uptake through an enhanced
degradation of IRS-1 (16, 128, 238, 247). Thus, it appears that mTOR signalling may be an
important step in the regulation of glucose uptake in skeletal muscle cells. However, the
specific role of mTOR signalling remains to be tested in healthy or Type II diabetic humans.
Study
Model
Method
Protein/AA source
Glucose metabolism
Signalling response
Dependent
on insulin
Peyrollier et al.
(2000) • L6 myotubes AA deprived muscle
cells were incubated
with AA.
Leu • N/a • AA ↑ p70S6K and ↓ GSK3 *
• Dependent on PI3K and
mTOR but not Akt *.
• No
Armstrong et
al. (2001)
• Cultured human
myoblasts
Myoblasts were
incubated in an amino
acid medium.
Mixed AA medium • AA medium ↑ GS
activation **
• Dependent on mTOR and
PI3K but not Akt **.
• AA ↑ p70S6K and ↓ GSK3
**
• No
Nishitani et al.
(2002)
• Isolated rat
soleus muscle
Isolated muscles
incubated in leucine.
Leu • Leu ↑ glucose uptake * • Glucose uptake with AA was
dependent on PI3K and
αPKC but not mTOR *.
• No
Doi et al.
(2003)
• Healthy rats
• C2C12 myotubes
Post-OGTT BCAA
feeding and glucose
uptake in myotubes.
Leu, Ile, Val or Con • Iso ↓ plasma glucose **
• Iso ↑ glucose consumption
*
• Dependent on PKC and
PI3K but not mTOR *
• No
Nishitani et al.
(2005) • Cirrhosis
induced rats
Post-OGTT BCAA
feeding and isolation of
soleus muscles.
Leu, Ile , Val or
control • Leu and Iso ↑ glucose
tolerance and uptake *
• Leu and Iso ↑GLUT4 and GS
activity *
• Leu phosphorylated p70S6K
and was dependent on mTOR
*
• No
Morifuji et al.
(2009) • L6 myotubes
• isolated rat
epitrochlearis
muscles
L6 myotubes and
isolated muscle
incubated with WPH
dipeptides.
WPH dipeptides
Iso+Leu.
• Dipeptides and Iso+Leu ↑
glucose uptake *
• Iso+Leu were dependent on
PI3K and αPKC **
• No
Morifuji et al.
(2009)
• Exercise trained
rats
Rats ingested glucose or
glucose plus protein
immediately post-
exercise.
Glu alone or with
WPH, CH, or BCAA • WPH ↑ muscle glycogen
levels at 2 hours post-ex *
• WPH ↑ phosphorylation of
Akt and PKC *
• Yes
Iwanaka et al.
(2009) • Isolated rat
epitrochlearis
muscle
Isolated muscle treated
with Leu following
insulin or contraction
Leu • Leu ↑contraction-induced
glucose uptake *
• Leu ↓ insulin-stimulated
glucose uptake *
• Leu ↑ phosphorylation of
p70S6K after contraction *
• Leu ↑ phosphorylation of
IRS-1 and ↓ Akt with insulin
*
• No
• Yes
OGTT; oral glucose tolerance test, BCAA; branched chain amino acids, Leu; leucine, Ile: isoleucine, Val; valine, GLUT4; glucose transporter protein,
GS; glycogen synthase, p70S6K
; ribosomal protein S6 kinase, mTOR; mammalian target of rapamycin, WPH; whey protein hydrolysate, PI3K;
phosphatidylinositol 3-kinase, PKC; atypical protein kinase C, Akt; protein kinase B, AA; amino acids, GSK3; GS kinase-3, IRS-1; insulin receptor
substrate-1, *; P < 0.05, **; P < 0.01.
Table 1.3 Studies investigating glucose metabolism and intracellular signalling in response to protein/amino acids on in rat and cell culture models.
General Introduction 45
1.8 Specific objectives of the thesis
This thesis describes a series of studies investigating the effectiveness of protein
nutrition on exercise performance, indices of recovery and the metabolic/molecular
adaptations to endurance and resistance exercise training. Chapter 2 describes a study in
which we determined whether the addition of protein to a carbohydrate beverage improved
cycle time-trial performance. Furthermore, we tested the hypothesis that carbohydrate plus
protein ingestion during exercise enhances recovery by measuring indirect markers of muscle
damage and muscle function. Chapters 3 and 4 focus on the influence of protein nutrition on
protein metabolism following acute endurance exercise and glucose metabolism following
acute resistance exercise. Chapter 3 describes a study in which we determined the synthetic
response of myofibrillar and mitochondrial proteins to carbohydrate plus protein ingestion
following prolonged cycling exercise. We simultaneously measured the phosphorylation of
intracellular signalling proteins implicated in translation initiation and elongation phase of
protein synthesis, in an attempt to provide a molecular mechanism to explain the divergent
response of specific protein fractions to post-endurance exercise protein ingestion. Chapter 4
describes a study in which we first determined the impact of a single bout of resistance
exercise on the glycemic response to an oral glucose load at 24 hours post-exercise. Second,
we tested the hypothesis that co-ingesting protein with the oral glucose load would augment
resistance exercise-induced improvements in glycemic control. During this experiment, we
utilized dual isotopic tracer methodology to measure endogenous and exogenous glucose
kinetics in combination with muscle biopsies to determine the underlying intracellular
mechanisms regulating glycemic control following resistance exercise and protein ingestion.
Chapter 5 discusses the results of the studies described in the previous chapters, providing
an overview of the main conclusions. The practical implications of the research are discussed
and recommendations for future research are provided.
General Introduction 46
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Chapter 2. 68
Leigh Breen, Kevin. D. Tipton., Asker. E. Jeukendrup.
School of Sport and Exercise Sciences, University of Birmingham, Birmingham, United
Kingdom.
Running title: Carbohydrate-protein beverages for athletes
Address for Correspondence:
Asker E. Jeukendrup, Ph.D.
The University of Birmingham
School of Sport & Exercise Sciences
Edgbaston; Birmingham
B15 2TT
Phone: +44 121 414 4124
Fax : +44 121 414 4121
Email: [email protected]
CHAPTER 2
NO EFFECT OF CARBOHYDRATE-PROTEIN ON CYCLING PERFORMANCE
AND INDICES OF RECOVERY
Chapter 2. 69
2.1 Abstract
Purpose: The aim of this study was to determine whether adding protein to a
carbohydrate beverage would improve late-exercise cycle time-trial performance over
carbohydrate alone. Furthermore, we examined the effects of co-ingesting protein with
carbohydrate during exercise on post-exercise markers of sarcolemmal disruption and the
recovery of muscle function. Methods: In a double-blind, cross-over design, 12 trained male
cyclists performed 120 min of steady-state cycling (SS) at ~55% V&O2max followed by a
time-trial lasting ~1 h (TT). At 15 min intervals during SS exercise participants consumed
either a carbohydrate (CHO) or carbohydrate-protein (CHO+Pro) beverage (providing 65
g·hˉ1 carbohydrate; or 65 g·hˉ
1 carbohydrate plus 19 g·hˉ
1 protein). Twenty-four hours
following the onset of the SS cycle, participants completed a maximum isometric strength
test. At rest and 24 h post-exercise, a visual analogue scale (VAS) was used to determine
lower-limb muscle soreness and blood samples were obtained for plasma creatine kinase
(CK) concentration. Dietary control was implemented 24 h prior to and during the time-
course of each trial. Results: Average power output sustained during TT was similar for
CHO and CHO+Pro with no effect of treatment on time-to-complete the time-trial (60:13 ±
1:33 min:sec and 60:51 ± 2:40 min:sec for CHO and CHO+Pro, respectively). Post-exercise
isometric strength significantly declined for CHO (15 ± 3%) and CHO+Pro (11 ± 3%)
compared with baseline (486 ± 28 N). Plasma CK concentrations and VAS soreness
significantly increased at 24 h post-exercise, with no difference between treatments.
Conclusions: The present findings suggest CHO+Pro co-ingestion during exercise does not
improve late-exercise time-trial performance, ameliorate markers of sarcolemmal disruption
or enhance the recovery of muscle function at 24 h post-exercise over carbohydrate alone.
Chapter 2. 70
Key words: Time-trial, endurance exercise, sarcolemmal disruption, muscle function.
Chapter 2. 71
2.2 Introduction
The quality and quantity of nutritional intake plays a critical role in response to training
and in athletic performance. It is well established that carbohydrate (CHO) ingestion during
moderate to high-intensity exercise can prolong endurance capacity (7). Evidence has since
emerged to suggest that the ingestion of multiple transportable carbohydrates can increase
exogenous carbohydrate oxidation and improve endurance performance, over and above the
effect of a single carbohydrate (8, 14). Recently, the addition of protein to a carbohydrate
containing beverage (CHO+Pro) during exercise has been reported to prolong endurance
time-to-exhaustion (TTE) in the range of 13-36% (13, 30, 31). In contrast, other well
controlled studies did not report such an ergogenic effect of CHO+Pro on time-trial
performance (25, 35). Recently, Saunders and co-workers (32) suggested the addition of
protein to carbohydrate specifically improved late-exercise time-trial performance. Thus, the
efficacy of adding protein to carbohydrates for endurance performance remains unclear.
A number of methodological differences between studies make it difficult to discern
whether any benefits observed for CHO+Pro are the result of a protein-mediated effect. First,
there are discrepancies in the way exercise performance is assessed for CHO and CHO+Pro
treatments. The ecological validity of TTE protocols is limited, because endurance athletes
do not compete in events that require sustaining a fixed power output for as long as possible.
Moreover, claims that CHO+Pro improves late-exercise performance were based on a time-
trial protocol that was not specifically designed to record power output and measure late-
stage exercise performance (32). Secondly, additional energy consumed when protein is
added to a carbohydrate containing beverage may explain the performance benefits observed
by others. If carbohydrate is consumed at a rate considered optimal for exogenous
Chapter 2. 72
carbohydrate oxidation (16), the role of protein for improving endurance performance may be
negligible (35). Finally, the control of external variables (or in some cases lack thereof) has
varied greatly between previous studies. Investigations attempting to implement a strictly
controlled exercise environment reported no effect of protein co-ingestion for time-trial
performance (25, 35).
The majority of studies examining the effect of carbohydrate-protein co-ingestion have
focused on the impact on endurance performance. Several authors suggest that consuming
CHO+Pro during and following endurance exercise improves recovery, defined as the
recovery of endurance capacity (30, 37) and muscle function (33, 34). Reductions in plasma
creatine kinase (CK) concentrations (20, 21, 28, 30, 31, 33, 34) and muscle soreness (20, 21,
28, 33), are thought to play a key role in aiding the recovery process. However, evidence to
support an effect of additional protein for improving recovery is inconsistent. Others have
found no difference in the post-exercise rise in plasma CK (5, 10), muscle soreness (5, 21) or
the recovery of endurance capacity (4) for CHO+Pro. Thus, the effectiveness of additional
protein for enhancing recovery is unclear, perhaps due to methodological and design
differences among studies, including variable amounts of control. These issues require further
investigation.
The primary aim of the present study was to investigate the efficacy of adding protein
to carbohydrate for improvement of late-exercise performance. Unlike previous studies, we
aimed to supply a recommended dose of carbohydrate during cycle exercise to determine
whether the addition of protein improved cycle time-trial performance in a controlled
exercise environment, when preceded by a standardized steady-state ride. Second, we aimed
to determine the effect carbohydrate-protein beverages on indices of recovery, 24 h following
Chapter 2. 73
time-trial cycle exercise by measuring plasma CK concentration, muscle soreness and muscle
function. Participant diet and physical activity were stringently controlled for the 48 h trial
duration to ensure participants were in a similar state of energy balance and fuel repletion.
2.3 Methods
2.3.1 Participants
Twelve trained male cyclists were recruited through advertisements, from local clubs
(subject characteristics are shown in Table 2.1). Only individuals who undertook 2 or more
training sessions per week of 1 – 5 h duration were eligible to participate. Data was collected
over a 4-month period, such that all participants were all in a similar phase of their training
cycle. All trials were completed within a 3-week period with no more than 14-days between
trials and the purpose and methodology of the study were clearly explained to participants.
All participants signed an informed consent and a completed a general health questionnaire
prior to taking part in the study. The experimental protocol was approved by the School of
Sport and Exercise Sciences Safety and Ethics Subcommittee.
2.3.2 Experimental Design
The study design was counter-balanced, cross-over and double-blind. Maximal oxygen
uptake and muscle function were assessed at baseline. Following baseline testing participants
performed two trials, during which they completed a 120 min steady-state cycle, consuming
either a carbohydrate (CHO) or carbohydrate-protein (CHO+Pro) treatment beverage.
Following the 120 min steady-state ride at 50%Wmax, participants were then asked to
complete a time-trial lasting ~1 h (Figure 2.1). Participants returned after a 7-14 days and
repeated the protocol whilst consuming the alternate treatment beverage. For example if the
participant consumed CHO+Pro during trial 1, they consumed the CHO treatment during trial
Chapter 2. 74
2. Markers of sarcolemmal disruption were obtained immediately prior to and 24 h post-
exercise and the recovery of muscle function was assessed 24 h post-exercise. All tests were
conducted in a fasted state at the same time-of-day to minimize any circadian variance.
2.3.3 Baseline Testing
Body Mass: A digital scale was used to determine body weight to the nearest 0.1kg.
Each participant was weighed in their cycling clothing without shoes. This was repeated
prior to each of the two testing visits to ensure body weight remained similar throughout.
Maximal Power Output: Prior to completing the treatment trials, maximal oxygen
uptake (V&O2max) and maximal power output (Wmax) were determined using an incremental
cycle test to exhaustion on an electrically braked cycle ergometer (LODE Excalibur Sport V
2.0 Groningen, Netherlands). The test consisted of a 3 min warm-up at 95 W, followed by an
increase of 35 W every 3 min until exhaustion, at a self-selected cadence. The seat position,
handlebar height and orientation used during testing were recorded and replicated on
subsequent visits to the laboratory. All cycle tests were conducted in thermo-neutral
conditions (23°C, 40% relative humidity). Wmax values were used to determine the
workload for the pre-loaded time-trial. Heart rate (HR) was measured continuously by using
Age
(Yrs)
Weight
(kg)
Experience
(y)
BMI
(kg·mˉ 2)
VO2max
(mL·kgˉ1·minˉ
1)
Wmax
(W)
Mean
27
70.5
7.1
22.4
62.7
350
SD 8 5 1.5
2.6
6.3
34
Table 2.1 Participant characteristics at baseline.
Data are presented as means ± SD
Chapter 2. 75
telemetry using HR monitor (Polar S625X; Polar Electro Oy, Kempele, Finland). V&O2 was
considered maximal if 3 of the 4 following conditions were met: 1) a plateau in V&O2 with
further increasing workloads (an increase of < 2 mL·kgˉ1·minˉ
1); 2) a HR within 10
beats/min of the age predicted maximum (220 bpm - age); and 3) a respiratory exchange ratio
(RER) of >1.05; 4) a rate of perceived exertion (RPE) greater than 17.
Metabolic & Physiological Measures: Expired O2, CO2 and RER, were measured
during the last 60 sec of each stage and ~60 sec prior to the cessation of exercise to determine
V&O2 and V&CO2. Participants indicated to the investigator the point at which they felt they
were only able to exercise for one more minute. These data were obtained using Douglas gas
collection bags (Plysu, Milton Keynes, UK), with air expired through a 2-way breathing
valve connected to the bag. Bags were analyzed using a gas analyzer (Servomex 1440, East
Sussex, UK), which was calibrated before each analyses using medical-grade gasses of
known concentrations (British Oxygen Company, UK). The RPE was obtained using the 6-20
Borg scale.
Assessment of Maximal Isometric Strength: Twenty-four hours after each time–trial, a
Tornvall chair (22) was used to determine maximal isometric strength (MIST). These values
were compared with a single rested MIST test conducted at baseline, to determine the post-
exercise loss of isometric strength. Both voluntary and stimulated isometric contractions of
the quadriceps femoris were determined, in the non-dominant leg. A series of known weights
was applied to a strain gauge and the resulting voltage output was recorded daily for
conversion into force. Participants were seated with the knee at 90o, which was fixed in place
by a cord fastened round the ankle. The cord was attached to a strain gauge interfaced with a
computer, on which the resultant force was displayed and recorded. Two arm straps held the
participant in place in the chair and minimize any extraneous movement. Damp electrodes
(13 x 8 cm) were secured proximally and distally to the anterolateral thigh. At baseline,
Chapter 2. 76
maximal electrical stimulation was determined using a twitch overlay programme;
progressively larger voltages (mA) were applied until two produced the same force. Once this
was determined participants received a percutaneous electrical stimulation three times before
being encouraged to extend their knee as hard as possible. Three electrical stimulations were
superimposed while contraction was occurring to ensure that each effort was maximal. This
procedure occurred three times during each trial with participants resting for 90 seconds
between efforts. The highest value was recorded as the participant’s maximal voluntary
contraction.
2.3.4 Blinded Trials
Two-hour Pre-load Cycle: No less than 7 days after baseline testing, participants
underwent familiarization of the time-trial whilst consuming water only. For each trial,
participants reported to the Human Performance Laboratory in a fasted state (06.00 – 07.00)
having undergone 24 h of dietary control prior to this visit. Participants completed 120 min
of constant pre-load ergometer cycling at a work-load 50% of their previously determined
Wmax (175 ± 5 W). Participants were instructed to maintain a comfortable cadence above 60
revolutions per minute (rpm).
One-hour Time-trial: Upon completion of the 120 min pre-load cycle the ergometer
was set in the linear mode and participants were asked to complete an energy based target
amount of work at 70% of their pre-determined Wmax. The total amount of work for a 1 h
time-trial (880 ± 27 kJ) was calculated according to the formula derived from Jeukendrup et
al. (15). A linear factor, calculated as work-rate at 70% Wmax divided by (90rpm2), was
entered into the ergometer controller. During the time-trial, participants received no verbal or
visual feedback regarding performance time or physiological measures. Participants had
access to a computer screen indicating the amount of the total energy target they had
Chapter 2. 77
completed. Trials were performed in a small testing laboratory with a screen placed behind
each participant to minimize distraction. Air conditioning and a fan were used when
requested by participants, for which the settings were recorded and replicated during the
second trial.
Metabolic & Physiological Measures: Oxygen uptake, CO2 production and RER data
were collected for 2 min intervals every 30 min of the 120 min pre-load cycle to determine
the intensity of the workload. Heart-rate and RPE were obtained also to confirm a steady
state was maintained prior to the time-trial. During the time-trial HR, RPE and average power
output were recorded at 25, 50 and 75% of the total energy target (kJ). These same measures
were also obtained ~10 seconds prior to completion of the trial.
Assessments of Muscle Function and Force Production: Twenty-four hours after the
start of the pre-loaded cycle trial (06.00 – 07.00), participants were asked to return to the
laboratory in a fasted state, to undergo a MIST as described previously.
Visual Analogue Scale of Muscle Soreness: Using a 50mm line with word descriptors at
each end reading ‘no soreness’ and ‘extreme soreness’, participants marked on the line, the
point that they felt represented their current degree of general muscle soreness in their non-
dominant leg. The distance to the mark was then measured in millimeters. VAS soreness was
also rated when the knee was both flexed and extended. Average VAS soreness is reported
here as it closely represents general, flexed and extended soreness values. A VAS was
completed in the morning prior to each time-trial and 24 h following the onset of the pre-load
cycle upon return to the laboratory prior to the MIST. The VAS scale has previously been
shown to be a reliable device in measuring acute pain (6).
Blood Analysis: Upon arrival for each time-trial, a cannula (IV Venflon 20GX32mm)
was inserted into the antecubital vein of the forearm. A two-way stopcock (Medex, Monsey,
NY) was applied and opened to collect a 10mL sample at rest. A further blood sample was
Chapter 2. 78
taken upon each participants return to the laboratory for the MIST on day 2 (24 h following
the onset of the pre-load cycle). Samples were centrifuged at 3000rpm at 4°C to allow the
extraction of blood plasma for further analysis. All plasma samples were processed and
stored frozen at -80°C. The exact time-points of all sample collections were recorded and
repeated during the second blinded treatment. Enzymatic analysis of CK concentration (CK
NAC CP, ABX diagnostics, UK) was performed in duplicate at each time-point using a semi-
automated analyzer (COBAS MIRA S-plus, ABX, UK).
2.3.5 Treatment Beverages
The two experimental trials were a 6% maltodextrin carbohydrate beverage (65 g·hˉ1;
CHO) and a carbohydrate + protein (CHO+Pro) beverage containing 6% maltodextrin (65
g·hˉ1) and 1.8% protein hydrolysate (19 g·hˉ
1). Treatment beverages were counter-balanced
throughout the study to minimize any influence of order, with 6 participants receiving CHO
first and 6 receiving CHO+Pro first. Beverages were served in non-see-through bottles, with
a different flavor used in each trial to minimize the risk of taste comparisons between
beverages. During pilot testing, a group of cyclists were able to successfully identify the
beverage composition when flavors were the same. Flavors were added using 3g of non-
energetic sweetened drink mix. Participants consumed 270mL every 15 min of the 120 min
steady state pre-load, beginning at the onset of the ride (1080mL per/hour). An investigator
handed the participant one container at a time and verbally encouraged them to drink at an
appropriate rate. Water was consumed ad libitum during the time-trial with the amount
recorded (~430 ± 26mL) and administered in the same volume during the second trial.
Beverages were prepared by an independent investigator who had no part in the data
collection of the study.
Chapter 2. 79
Figure 2.1 Schematic diagram of the experimental protocol. Metabolic measures include
V&O2, V&CO2 and RER. Physiological measures include HR, RPE and average power output.
Time values are in hours.
2.3.6 Diet & Exercise Control
Participant diet was standardized for the 48 h duration of each treatment. This ensured
that nutrient intake was controlled for 24 h prior to each time-trial and over the 24 h course of
each time-trial day, prior to the assessment of muscle function the following morning. In-
between baseline testing and each blinded trial, participants completed a 3-day food diary,
representative of their average week. A questionnaire of food preferences was also completed
by participants. Using an on-line diet planner, each of the 3-days was logged and the energy
and nutrient intake estimated. An average of the 3-days was combined with the participant’s
weight to calculate the total energy content of the diet. The standardized diet was matched to
each participants habitual daily energy intake and contained 8g·kgˉ1
BM carbohydrate and
1.6g·kgˉ1
BM protein with the remainder of energy derived from fat. The macronutrient
composition of the standardized diet was not significantly different from participants’
habitual diet (Table 2.2). Participants were instructed to consume only the food provided for
Beverage (270mL)
Blood sample & VAS
Metabolic &
Physiological Measures
-24 0 1 2 ~3 24 25
Dietary control
50% Wmax 70% Wmax MIST
Chapter 2. 80
them over the two-day testing period. The same two-day food parcel was provided to each
participant prior to the second trial. Participants were instructed to maintain normal volume
and intensity of training throughout the course of the study but to refrain from training for 48
h prior to each testing phase.
2.3.7 Statistical Analysis
Data are reported as mean ± SEM, unless otherwise stated. All data were analyzed by
two-way analysis of variance for repeated measures (Group x Time). Significant differences
between means were determined using SPSS (Version 15.) Bonferroni adjustment was
applied to post hoc means-comparison tests. Differences were considered significant at P <
0.05. In keeping with recent trends in inferential statistics, we made magnitude based
inferences about the ‘true’ values of the effect of treatment on outcomes by expressing the
uncertainty as 90% confidence limits (CL) and by calculating and interpreting chances that
the true effect was beneficial or detrimental. For time-trial performance data a substantial
effect was derived from 0.5 times (11) the average of estimates for the typical error of
measurement for time-trial performance and power output. For all other biochemical and
psychometric variables, the smallest standardized (Cohen) difference in the mean (0.20 times
the between-subjects standard deviation for the control group) was used to identify the
magnitude of the smallest substantive effect. Based on an analysis of the confidence limits
and P values using a published spreadsheet (2), the likelihood of a substantial benefit or
detriment, increase or decrease, for an outcome was qualified as follows: <1%, almost
certainly not; 1–5%, very unlikely; 5–25%, unlikely; 25–75%, possible; 75–95%, likely; 95–
99%, very likely; and >99%, almost certain (9); otherwise, an effect was deemed unclear or
inconclusive if the confidence interval overlapped the thresholds for positive and negative
substantial effects by >5%. When the mean and >50% of the confidence interval lie within
Chapter 2. 81
the threshold for a substantial effect, the effect is qualified as trivial. P values were provided
for an inferential comparison for the main experimental outcomes only.
2.4 Results
2.4.1 Time-trial performance
Mean power output during the time-trial was not significantly different between
treatments (247 ± 11 W for CHO and 247 ± 13 W for CHO+Pro, respectively). Power output
was reduced at 75%, compared with 25 and 50% of time-trial completion (Figure 2.2);
however, average power outputs at these time-points were not different between treatments.
As a result there was also no significant difference in time-to-complete the time-trial for
CHO or CHO+Pro (Figure 2.3). Inference based statistics determined the effect of CHO+Pro
compared with CHO on time-to-complete the trial was unclear (P = 0.81). Heart rate and
RPE were significantly elevated at completion of time-trial target compared with 25, 50 and
75% completion (P < 0.001) with no between-group differences at any time-point between
CHO and CHO+Pro (Table 2.3).
2.4.2 Steady-state exercise
There was no between-group difference in heart rate and V&O2 at 0, 30, 60, 90 and 120
min of the 120 min pre-load cycle. Average HR for CHO and CHO+Pro was 141 ± 12 and
142 ± 12 bpm respectively (Table 2.3). Average V&O2 (34.9 ± 3.7 mL·kgˉ1·minˉ
1 for CHO and
34.3 ± 4.2 mL·kgˉ1·minˉ
1 for CHO+Pro) and RER (0.88 ± 0.01 for both) were not different
between treatments at any time-point. Metabolic data indicated participants were cycling at
~55% V&O2max. Throughout the steady state ride RPE was not significantly different between
treatments (Between-subject range 10 – 15).
Chapter 2. 82
Table 2.2 Comparison of habitual and standardized diet
Carbohydrate Protein Fat
Habitual diet
(g)
570.3 ± 41.9
104.8 ± 12
74.8 ± 41.2
(g·kg-1
BM) 8.1 ± 0.5 1.5 ± 0.1 1.1 ± 0.6
Standardized
diet
(g) 565.2 ± 39.4 113.1 ± 7.8 73.4 ± 35.6
(g·kg-1
BM) 8.0 ± 0 1.6 ± 0 1.0 ± 0.5
2.4.3 Isometric knee extensor strength
A significant time effect revealed that knee extensor MIST declined 24 h after the onset
of each trial compared with baseline MIST (15 ± 3% for CHO and 11 ± 3% for CHO+Pro; P
Heart-rate (bpm)
Rate of perceived exertion (6-20)
Trial
Completion
CHO
CHO+Pro
CHO
CHO+Pro
25%
165 ± 3
166.8 ± 2.6
16.3 ± 0.5
15.8 ± 0.8
50%
167± 4
168.1 ± 2.8
17.5 ± 0.7
16.6 ± 0.7
75%
165 ± 3
169.2 ± 2.3
18.0 ± 0.7
17.2 ± 0.7
100%
a 180 ± 3
a 180.6 ± 2.1
a 19.4 ± 0.6
a 19.2 ± 0.8
Table 2.3 Physiological data at 25, 50, 75, and 100% of time-trial completion.
*: Significant increase compared with 25, 50 and 75% completion time-points (P < 0.001)
indicated by a. Data are presented as means ± SEM.
Participant daily habitual energy intake was 3373.3 ± 349.1 kcal. Habitual energy intake was
maintained during standardized dietary control. Macronutrient intake (g·kg-1
BM) was not
significantly different between habitual and standardized diets (P < 0.05). Values are presented as
means ± SD.
Chapter 2. 83
< 0.002); with no significant between-group difference (Figure 2.4). Inference based
statistics revealed the effect of CHO+Pro over CHO on the post-exercise recovery of knee
extensor MIST was most likely trivial (P = 0.28).
2.4.4 Visual analogue scale of muscle soreness
A significant time effect (P < 0.05) revealed VAS soreness increased from 4.8 ± 1.2 to
14.3 ± 4.3mm for CHO and 5.9 ± 1.9 to 14.3 ± 4.8mm for CHO+Pro, 24 h after the onset of
each trial (Figure 2.5). No between-group difference in VAS soreness was evident at 24 h
post-exercise. Inference based statistics indicated no clear difference in the extent of VAS
muscle soreness between-groups (P = 0.91).
2.4.5 Plasma creatine kinase concentration
A significant time interaction (P < 0.002) revealed plasma CK was significantly
increased for CHO and CHO+Pro at 24 h post-exercise (Figure 2.6). No between-group
difference in plasma CK concentrations was evident at 24 h post-exercise (P > 0.05).
Inference based statistics revealed the effect of CHO+Pro compared with CHO on post-
exercise CK concentrations was most likely trivial (P = 0.53).
2.4.6 Treatment Blinding
Seven of the 12 participants completed the time-trial more quickly during their first
treatment visit, suggesting there was no learning effect between trials. Five of the 12
participants correctly identified the beverage order during testing. Of the 5 participants who
identified the beverages correctly, only 2 performed faster with CHO+Pro suggesting our
attempts to minimize treatment bias were successful. Seven of the 12 participants, performed
Chapter 2. 84
better when consuming a CHO beverage, with the remaining 5 completing the task more
quickly with CHO+Pro.
Time-to-Completion (min)
0 45 50 55 60 65
CHO
CHO+Pro
Figure 2.2 Average power output measured at quarterly completion of time-trial target. a,
indicates significant reduction in power output vs. all prior time-points. b, indicates significant
increase in power output vs. 75% completion (P < 0.05). Data are presented as means ± SEM.
Figure 2.3 Time-trial time-to-completion. Data are presented as means ± SEM.
% TT Completion
25% 50% 75% 100%
Po
we
r O
utp
ut
(W)
0
230
240
250
260
270 CHO
CHO+Pro
a
a b
Chapter 2. 85
Figure 2.4 Maximum isometric force production of the knee extensors at baseline and 24 h post-
exercise. a, indicates significant reduction from baseline (P < 0.05). Data are presented as means
± SEM.
Figure 2.5 Average visual analogue scale of perceived muscle soreness at rest and 24 h post
exercise. Muscle soreness scored on a scale of 0 (no soreness) to 50 (extreme soreness). a,
indicates significant increase from rest (P < 0.05). Data are presented as means ± SEM.
Baseline CHO CHO+Pro
Iso
me
tric
Fo
rce
(N
)
0
100
200
300
400
500
600
a a
Rest Post 24 h
VA
S S
ore
ne
ss (
mm
)
0
5
10
15
20CHO
CHO+Pro
a a
Chapter 2. 86
2.5 Discussion
The present study is the first to our knowledge, to determine the effect of a CHO+Pro
beverage on late-stage cycle time-trial performance and post-exercise indices of recovery.
Here we show that the addition of protein to a moderate carbohydrate dose did not improve
late-exercise time-trial performance when beverages were consumed during a steady-state
pre-load cycle. Furthermore, there were no differences in power output between treatments at
any stage of the time-trial. Finally, the addition of protein to a carbohydrate containing
beverage did not improve any of the measured indices of post-exercise recovery. Thus, our
results do not support the notion that carbohydrate-protein co-ingestion during exercise
enhances late-exercise performance or post-exercise recovery over to carbohydrate alone.
In contrast to the present findings, several investigations suggest that the addition of
protein to carbohydrate improves endurance performance (13, 30-32). In many cases the
Figure 2.6 Plasma creatine kinase (CK) concentration at rest and 24 h post-exercise. a, indicates
significant increase from rest (P < 0.05). Data are presented as means ± SE.
Rest Post 24 h
Pla
sm
a C
K (
U/L
)
0
100
200
300
400 CHO
CHO+Pro
a
a
Chapter 2. 87
discrepancies between studies may be due to differences in the exercise test used to assess
performance. Studies suggesting improved performance actually measured time-to-
exhaustion (13, 30, 31), not performance per se. Currell and Jeukendrup (9) highlighted the
fact that the ecological validity of exhaustive exercise protocols is limited, because endurance
athletes do not compete in events that require sustaining a fixed power output for as long as
possible. Furthermore, the precision of time-to-exhaustion is reportedly in the range of 26%
(15). On the contrary, time-trial cycle tests have been found to be highly reproducible and
sensitive to small changes in exercise performance (15, 18, 26). Our observation of no
improvement in time-trial performance for CHO+Pro is consistent with other studies
measuring overall time-trial performance (25, 35). It should be noted that the final
measurement of performance is influenced not only by the exercise test, but by control
measures employed and subject feedback during exercise. Knowledge of parameters such as
time elapsed; distanced travelled and heart rate during exercise, may compromise the
blinding of treatments, creating a placebo effect (23), particularly when additional protein is
consumed which is often difficult to mask. Early studies did not report the control of these
conditions (13, 28, 30-32, 34). Further to the well controlled study protocol we used,
participant diet was assessed and standardized, whilst physical activity was minimized to
ensure participants were in a similar state of energy balance and fuel repletion prior to and
during each treatment trial. Thus, when performance, rather than endurance capacity is
measured in a controlled environment, additional protein does not seem to be advantageous
for performance.
Recently, the importance of additional protein on more specific aspects of endurance
performance has been touted. In particular, the notion that additional protein is important for
enhancement of performance late in exercise has been put forward (32). Despite no difference
in 60km time-trial performance between CHO and CHO+Pro treatments, ingestion of
Chapter 2. 88
additional protein resulted in improved performance during the final stages of the time-trial
(32). Whereas this result can be interpreted as important for endurance athletes, it should be
emphasized that overall performance was not improved, thus minimizing the importance of
any late exercise improvement. Given that overall performance was not different, the shorter
time taken to complete late-exercise stages with CHO+Pro may have been due to a slower
completion of earlier exercise stages, or greater variability in the earlier section of the time-
trial relative to more reliable late-exercise performance. Whereas, these results contrast with
our findings, the differences may be due to the fact that Saunders and colleagues (32) did not
design their study to specifically examine the impact of additional protein on late-stage
performance. The pre-loaded cycle time-trial used in the present study was designed to record
power output and determine late-exercise time-trial performance and has been validated in
previous studies from our laboratory (9, 15). In support of our findings, a recent study by
Osterberg and colleagues (25) found no enhancement of late-exercise time-trial performance
preceded by a 120 min steady-state ride, when protein was added to carbohydrate. Accurate
determination of late-exercise performance requires that the exercise intensity of the prior
bout should be standardized and power output reported. Thus, it seems that when properly
controlled, late-stage exercise performance is not improved by the addition or protein to
carbohydrate during exercise.
Furthermore, lack of a definitive physiological mechanism also contributes to doubt of
the importance of adding protein to carbohydrate during exercise. Several mechanisms have
been theorized (29), but only one has any support in studies. Koopman and colleagues (17)
showed that ingesting CHO+Pro during ultra-endurance exercise resulted in two-fold greater
protein oxidation compared with CHO. Thus, studies in which extended time-to-exhaustion
was found, suggest that the addition of protein may alter substrate utilization, potentially
sparing muscle glycogen (13, 30, 31). However, carbohydrate intake in these studies was too
Chapter 2. 89
low (<50 g·hˉ1) to attain peak exogenous oxidation rates (16) and the protein plus
carbohydrate provided more energy than the carbohydrate alone. Investigations in which the
total energy content of CHO and CHO+Pro beverages was matched, have found no difference
in time-to-exhaustion (28, 34). Thus, the benefit of adding protein to carbohydrate likely is
due to the additional energy delivered by protein as opposed to a protein per se. We show the
addition of protein (19 g·hˉ1) to a carbohydrate dose (65 g·hˉ
1) that meets the recommended
upper-limit to attain peak exogenous carbohydrate oxidation rates, did not improve late-
exercise time-trial performance despite increasing the total beverage energy load by 29%. To
our knowledge, no other purported mechanism for the enhancement of performance by
additional protein has been demonstrated at this time.
In addition to the enhancement of endurance performance, adding protein to
carbohydrate has been promoted to enhance recovery. Previous studies have shown
CHO+Pro beverages prolong subsequent time-to-exhaustion in the range of 40 and 55% (30,
33, 37). Others have found the addition of protein to carbohydrate results in small increases
in the recovery of muscle function, on the order of 1-2 knee extension lifts (34) and 1-2cm in
vertical jump height (34). Whereas these data suggest the addition of protein to carbohydrate
improves the recovery of performance and function, there are many factors that differ
between studies, thus the picture is less than clear. It is difficult to determine whether these
benefits were influenced by the timing of beverage ingestion (prior to, during, or post-
exercise), the way in which recovery was measured, or the time given between exercise and
the assessment of recovery. We chose to measure maximal isometric strength at 24 h post-
exercise because of its practical relevance (1). Athletes often repeat their training or compete
24 hours after an exercise bout and therefore recovery of muscle function and exercise
performance would be important in this time frame. In concert with two recent studies (5,
10), we showed that the co-ingestion of carbohydrate and protein, does not improve the
Chapter 2. 90
recovery of isometric strength. Others have failed to show improvements in recovery as based
on improved subsequent performance assessed in various methods (4, 10, 20, 21, 28) . Thus,
the impact of adding protein to carbohydrate on recovery from intense exercise must still be
considered to be somewhat equivocal.
Studies that report improvements in the recovery of endurance capacity typically
measured these changes several hours following protein ingestion (30, 37). The ingestion of
protein is known to stimulate muscle protein synthesis (12) resulting in positive net whole-
body protein balance following endurance exercise (19), which has been suggested to
enhance recovery through the repair and remodeling of damaged proteins (20, 30, 31).
However, the turnover of myofibrillar proteins is relatively slow, on order of days to weeks
(27). Thus, it is difficult to comprehend how this process may influence changes noted in just
a few hours (30, 37). So, if recovery is enhanced by adding protein to carbohydrate, it is
unlikely to be due to the improved net muscle protein balance from the protein.
Whereas recovery may be defined by the ability to reproduce an optimal level of
performance, several investigations have sought to measure the effect of CHO+Pro on the
other markers thought to be important to the recovery process. Studies have shown that
CHO+Pro beverages reduce plasma CK, a putative marker of sarcolemmal disruption and
muscle soreness, (20, 21, 28, 31, 32). Others have found that lower CK concentrations and
lower ratings of muscle soreness accompany improvements in the recovery of time-to
exhaustion (30, 33) and vertical jump height (33). Consequently, previous studies suggest
that protein co-ingestion may play an important role in reducing plasma CK and muscle
soreness, thus improving recovery. However, the changes in plasma CK when protein is
added to carbohydrate are often minimal (20, 28, 31, 32, 34) and the inter-individual variation
is very large, making it difficult to determine the physiological significance. Moreover, the
plasma CK and muscle soreness values found in our study are in a similar range to those
Chapter 2. 91
reported previously, yet we show that CHO+Pro did not ameliorate post-exercise markers of
sarcolemmal disruption. A recent study from Betts et al. (5) used a strenuous bout of
intermittent shuttle running to induce a greater degree of sarcolemmal disruption than the
cycle time-trial used in our study. In support of our findings, the authors showed that the
addition of protein to a carbohydrate beverage did not ameliorate post-exercise plasma CK
and muscle soreness. Thus, it is not clear what impact, if any, ingestion of protein has on
recovery by ameliorating CK and soreness.
It is likely that the importance of plasma CK and muscle soreness for enhancement of
recovery has been over emphasized by studies that suggest the addition of protein to
carbohydrate attenuates these responses (20, 21, 28, 31, 32). At best, plasma CK and muscle
soreness should be considered putative markers of muscle damage. Studies have shown that
these indirect markers have a poor relationship with the loss of muscle function (24) and
direct markers of sarcolemmal disruption (3, 36). Moreover, ‘true’ treatment effects may be
masked by the inherent variability of the CK response (5, 24, 36). Further to this suggestion,
three participants in our study exhibited three-fold greater resting plasma CK response
compared with the group average when protein was co-ingested. Resting CK concentrations
for these same individuals were vastly reduced during the carbohydrate-only trial. Thus, our
results suggest a clear need to further investigate the impact of protein ingestion on recovery
from endurance exercise. It would seem that studying direct markers of sarcolemmal
disruption (biopsy or MRI techniques) in concert with post-exercise tests of muscle function
may provide clearer answers regarding the efficacy of CHO+Pro for improving recovery.
In conclusion, when energy intake is controlled and carbohydrate is ingested at rates
considered optimal for peak exogenous carbohydrate oxidation, the addition of protein does
not improve late-exercise cycle time-trial performance or recovery of muscle function at 24 h
post-exercise. The metabolic and physiological response to CHO+Pro may be dependent on
Chapter 2. 92
the mode, duration and intensity of the exercise bout and warrants further study. Based on our
findings and other carefully controlled studies (25, 35) there is currently no basis to
recommend CHO+Pro beverages to endurance athletes for performance enhancement or
improved recovery.
Acknowledgment
First and foremost the authors would like to thank the participants for their time and
effort. The authors would also like to acknowledge the experimental support of Matthew
Cocks and Hollie Williams. No funding was received for this study. There are no conflicts of
interests for any of the authors.
Conflict of Interest
There are no conflicts of interest for any of the authors. The results of the present study
do not constitute endorsement by ACSM.
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Chapter 3. 96
CHAPTER 3
THE INFLUENCE OF PROTEIN INGESTION FOLLOWING ENDURANCE
EXERCISE ON MYOFIBRILLAR AND MITOCHONDRIAL PROTEIN
SYNTHESIS.
Leigh Breen1, Andrew Philp
2, Oliver. C. Witard
1, Sarah. R. Jackman
1, Anna Selby
3, Ken
Smith3, Michael. J. Rennie
3, Keith Baar
2, Kevin. D. Tipton
1, 4
1School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK.
2Functional Molecular Biology Lab, Neurobiology, Physiology and Behaviour, University of
California Davis, US .3School of Graduate Entry Medicine & Health, University of
Nottingham, Derby, UK. 4Department of Sports Studies, University of Stirling, Stirling, UK.
Running title: Carbohydrate-protein beverages for endurance athletes
Address for Correspondence:
Kevin. D. Tipton, Ph.D.
The University of Stirling
Department of Sports Studies
Stirling, UK.
FK9 4LA
Email: [email protected]
Chapter 3. 97
3.1 Abstract
Purpose: The aim of the present study was to determine mitochondrial and
myofibrillar muscle protein synthesis (MPS) when carbohydrate (CHO) or carbohydrate plus
protein (C+P) beverages were ingested following prolonged cycling exercise. The
intracellular mechanisms thought to regulate MPS were also determined. Method: In a
single-blind, cross-over study, 10 trained cyclists (age 29 ± 6 yr, V&O2max 66.5 ± 5
mL·kgˉ1·minˉ
1) completed two trials in a randomized order. Subjects cycled for 90 min at
~77 ± 1% V&O2max before ingesting a CHO (25g of carbohydrate) or C+P (25g carbohydrate
+ 10g whey protein) beverage immediately and 30 min post-exercise. A primed constant
infusion of L-[ring-13
C6] phenylalanine began 1.5 h prior to exercise and continued until 4 h
post-exercise. Muscle biopsy samples were obtained to determine myofibrillar and
mitochondrial MPS and the phosphorylation of intracellular signalling proteins. Arterialized
blood samples were obtained throughout the protocol. Results: Plasma amino acid, plasma
urea and serum insulin concentrations increased following ingestion of C+P only.
Myofibrillar protein synthesis was greater for C+P compared with CHO (0.087 ± 0.007 and
0.057 ± 0.006 %·h-1
, respectively; P = 0.025). Mitochondrial protein synthesis rates were
similar for C+P and CHO. mTORSer2448
phosphorylation was greater for C+P compared with
CHO at 4 h post-exercise (P < 0.05). p70S6KThr389
phosphorylation increased at 4 h post-
exercise for C+P (P < 0.05), whilst eEF2Thr56
phosphorylation increased by ~40% at 4 h post-
exercise for CHO only (P < 0.01). Conclusions: The present study demonstrates that the
ingestion of C+P following prolonged cycling stimulates an increase in myofibrillar MPS.
These data indicate that the increase in myofibrillar MPS for C+P may be mediated through
the phosphorylation of p70S6K, downstream of mTOR, which in turn may release the
inhibition of eEF2 on translation elongation.
Key words: cycling, skeletal muscle, protein turnover, training adaptation.
Chapter 3. 98
Abbreviations: EE, endurance exercise; RE, resistance exercise; MPS, muscle protein
synthesis; C+P, carbohydrate plus protein; CHO, carbohydrate-only; V&O2, oxygen uptake;
mTOR, mammalian target of rapamycin; p70S6K, 70kDa S6 protein kinase; Akt, protein
kinase B; 4E-BP1, eukaryotic initiation factor 4E binding protein 1; eEF2, eukaryotic
elongation factor 2; PRAS40, proline-rich Akt substrate 40 kDa; p38 MAPK, p38 mitogen-
activated protein kinase; AMPK, AMP-activated protein kinase.
Chapter 3. 99
3.2 Introduction
Endurance (EE) and resistance exercise (RE) training regimens result in divergent
phenotypic adaptations. Whereas RE promotes muscle hypertrophy and an increase in
contractile force output (20, 29), EE training is characterized by an expansion of oxidative
capacity, brought about through an increase in the size and density of mitochondria (25, 47).
At the metabolic level, the adaptation to exercise is determined by summing the acute (22, 35,
48) transcriptional (24, 42) and translational responses (54) to each exercise stimulus and the
subsequent increase in synthesis of muscle proteins. Recently, Wilkinson and colleagues (54)
showed the response of myofibrillar and mitochondrial muscle protein synthesis (MPS) were
dependant on the type of exercise performed. Furthermore, the interaction of exercise and
protein nutrition varies between different protein fractions (38). Thus, it is important to
examine the response of different protein pools to exercise in order to determine whether a
particular nutritional intervention is having the desired effect.
Whereas the response of mixed MPS to different types of exercise has been
investigated (19, 41, 50), there is less information on the response of various proteins to
exercise and nutrition. It is widely recognized that ingesting protein potentiates the anabolic
effect of RE (45), seemingly due to the essential amino acid content (49). Recently, Moore et
al. (38) showed a differential response of myofibrillar and sarcoplasmic proteins to RE and
protein nutrition. However, very few studies have sought to determine the effect of protein
ingestion on MPS following EE. Levenhagen et al. (34) showed that adding protein to a
carbohydrate treatment (C+P) increased post-EE leg and whole-body protein synthesis and
this increase in synthesis was associated with net protein gain. However, it was unclear
whether the benefits observed were due to the protein per se, or an increase in total energy
intake. To remedy this, Howarth and colleagues (27) showed that C+P ingestion increased
Chapter 3. 100
mixed MPS compared with CHO treatments matched for total energy and carbohydrate
content. On the other hand, another recent study showed no increase in mixed MPS when
C+P was ingested following EE (19). Importantly, none of these studies (19, 27, 34) have
attempted to resolve the specific protein fractions contributing to changes in mixed MPS, or
the mechanism accounting for the adaptations. Therefore, specifically investigating the acute
response of mitochondrial or myofibrillar protein synthesis to C+P ingestion following EE
may resolve this issue.
Acute changes in transcription and translation that occur following exercise regulate
skeletal muscle protein turnover through a number of intracellular signalling proteins. The
mammalian target of rapamycin (mTOR) is a key regulator of translational control,
integrating environmental signals from nutrients and exercise to control cell growth (13). The
activation of mTOR signalling leads to the phosphorylation of downstream targets involved
in the mRNA translation initiation and elongation (6), e.g. p70 ribosomal protein S6 kinase-1
(p70S6K). In concert with an increase in mitochondrial MPS following EE, Wilkinson et al.
(54) showed an increase in the phosphorylation of signalling proteins in the mTOR–p70S6K
pathway. Furthermore, human (28) and rat studies (39) indicate that post-EE C+P ingestion
increases the phosphorylation of intermediates in the mTOR-p70S6K pathway. To date, no
study has characterized the response of signalling proteins to changes in myofibrillar and
mitochondrial MPS following post-EE protein ingestion.
Thus, the primary aim of the present study was to determine whether ingesting
additional protein with carbohydrate following prolonged cycling exercise modified the
synthetic response of mitochondrial and myofibrillar muscle proteins. The second aim of the
study was to elucidate the potential intracellular signalling mechanisms (in the mTOR-
Chapter 3. 101
p70S6K pathway) regulating the differential response of myofibrillar and mitochondrial MPS
following post-EE protein ingestion.
3.3 Methods
3.3.1 Participants
Ten well-trained, male cyclists were recruited from local clubs through advertisements.
The mean (±SD) age of the cohort was 29 ± 6 yr, body mass was 77 ± 6.5 kg, maximal
oxygen consumption was 66.5 ± 5 mL·kgˉ1·minˉ
1 and maximal power output was 383 ± 25
W. Only cyclists who undertook 2 or more training sessions per week of 1–5 h duration were
eligible to participate. Participants had 6.5 ± 3 yr of competitive cycling experience. All tests
were completed within a 4-week period with both treatment trials separated by 14 to 21-days,
with the exception of one participant who completed the second trial 32-days after the first.
The purpose and methodology of the study were clearly explained to the participants. All
participants gave their informed consent prior to taking part in the study and were deemed
healthy based on their response to a general health questionnaire. The experimental protocol
was approved by the Black Country Research Ethics Committee (Rec No: 08/H1202/130).
3.3.2 Study Design
Participants reported to the laboratory on 3 separate occasions. During the first visit
maximal aerobic fitness was determined. Approximately 2-weeks later participants
performed the first blinded trial in which they consumed a carbohydrate (CHO) or
carbohydrate-protein beverage (C+P; Lucozade Sport Recovery Powder, GlaxoSmithKline,
Brentford, UK). Briefly, during each trial participants performed a 90 min high intensity,
steady-state cycle before consuming CHO or C+P, immediately and 30 min following the
exercise bout. Mitochondrial and myofibrillar MPS were measured by incorporating isotopic
Chapter 3. 102
tracer infusion and muscle biopsy techniques. Participants returned to the laboratory for the
second blinded trial 14 to 21-days after the first trial, thereby serving as their own control.
Trial order was randomized and counter-balanced.
3.3.3 Preliminary Testing
Body Mass: A digital scale was used to determine body mass to the nearest 0.1kg. Each
participant was weighed in their cycling clothing without shoes on. Measurement of body
mass was repeated prior to each of the two testing visits to ensure body mass remained
constant throughout the study.
Maximal Power Output: Maximum oxygen uptake (V&O2max) and maximal power output
(Wmax) were determined using an incremental cycle test-to-exhaustion on an electrically
braked cycle ergometer (LODE Excalibur Sport V 2.0 Groningen, Netherlands). The test
consisted of a 3 min warm-up at 95 W, followed by an increase of 35 W every 3 min until
exhaustion, at a self-selected cadence. Breath-by-breath measurements were taken throughout
exercise, using an OxyCon Pro automated gas analysis system (Jaeger, Wuerzeburg,
Germany). The gas analyzers were calibrated using a 4.95% CO2 - 95.05% N2 gas mixture
(BOC Gases, Surrey, UK) and the volume transducer was calibrated with a 3L calibration
syringe. Heart rate (HR) was measured continuously via telemetry using a HR monitor (Polar
S625X; Polar Electro Oy, Kempele, Finland). V&O2 was considered maximal if 3 of the 4
following conditions were met: 1) a plateau in V&O2 with further increasing workloads (an
increase of < 2 mL·kgˉ1·minˉ
1); 2) a HR within 10 beats/min of the age predicted maximum
(220 bpm - age); 3) a respiratory exchange ratio (RER) of >1.05; and 4) a rate of perceived
exertion (RPE) greater than 17. The seat position, handlebar height and orientation used
during baseline testing were recorded and replicated on subsequent visits to the laboratory.
Chapter 3. 103
All exercise bouts were conducted in thermo-neutral conditions (21°C, 40% relative
humidity).
Dietary Analysis & Control: Participant diet was standardized for 48 h prior to each
treatment. During the preliminary testing phase, participants completed a 3-day food diary,
representative of their average week (2 weekdays and 1 weekend day). A questionnaire of
food preferences was also completed by participants. Using an on-line diet planner (Weight
Loss Resources), each of the 3-days was logged and energy and macronutrient intake was
estimated. In our study cohort the average total daily energy intake was ~2787 ± 164 kcal
(5.3 ± 0.4 g·kg-1
BM carbohydrate; 1.2 ± 0.1 g·kg-1
BM fat; 1.5 ± 0.2 g·kg-1
BM protein). The
food parcels given to each participant matched their habitual energy and macronutrient
intake. Participants were instructed to refrain from caffeine and alcohol and to consume only
the food provided for them over the two-days prior to arriving for each trial. Participants
were also asked to consume their final meal no later than 2200 h to ensure a 10 h fast prior to
measuring myofibrillar and mitochondrial protein synthesis. An identical two-day food parcel
was provided to each participant prior to the second trial.
Physical activity control: Participants were instructed to maintain their normal training
volume and intensity throughout the course of the study but to refrain from training for 48 h
prior to each treatment trial. To monitor physical activity between trials, participants were
asked to record all training 7-days prior to each trial. No differences were noted in training
volume in the 7-days prior to each trial.
3.3.4 Experimental trial protocol
Chapter 3. 104
Each participant was instructed to arrive at the Human Performance Laboratory at
0630 h after an overnight fast, where standard measures of height and weight were taken. A
cannula was placed into the forearm vein of one arm and a hand vein of the other. The
forearm cannula was used to infuse a stable isotopic tracer whilst the hand vein was heated
for frequent arterialized blood sampling. After a resting blood sample had been obtained,
participants then received a primed constant infusion of L-[ring-13
C6] phenylalanine (prime: 2
µmol·kg-1
; infusion: 0.05 µmol·kg-1
·min-1
; Cambridge Isotope Laboratories, MA, USA) to
determine skeletal muscle protein kinetics (described below). Approximately 90 min after the
start of the infusion, having rested in a supine position, participants were asked to complete a
90 min cycling exercise bout on a Lode Cycle Ergometer at a self selected cadence ≥ 60
revolutions per minute (rpm). The exercise bout consisted of a warm-up cycle at 50% Wmax
(189 ± 4 W) for 10 min followed by 80 min, initially at 75% Wmax (283 ± 6 W). V&O2, RER,
HR and RPE were recorded over 25-30 min, 55-60 min and 85-90 min of the exercise bout
(as described above). If participants indicated during exercise that the workload was too
difficult and they were unlikely to complete the full 80 min, workload was lowered in 5%
decrements to no less than 65% Wmax. Air conditioning and a fan were used when requested
by participants. The exact settings of the air conditioning/fan and the time of any change in
workload were recorded and replicated during the second trial. Throughout each trial
participants were allowed to drink water ad libitum. The amount of water consumed
throughout the course of the each trial was found to be similar (1556 ± 249mL for CHO and
1674 ± 233mL for C+P).
Muscle biopsy and blood sampling Using a 5-mm Bergstrom biopsy needle, two
muscle biopsies (~100-150 mg of muscle tissue per biopsy) were obtained from the same leg
during each trial. The order of biopsied leg was randomized and counterbalanced for each
Chapter 3. 105
trial. Prior to the exercise bout (~15min) under local anaesthetic (1% Lidocaine), the lateral
portion of one thigh was prepared for the extraction of a needle biopsy sample from the
vastus lateralis muscle. Biopsy incisions were made prior to exercise to allow the sample to
be obtained as quickly as possible after exercise (5 ± 1 min post-exercise). Immediately after
the post-exercise muscle biopsy was obtained, participants were asked to consume one of two
treatment beverages described below. Four hours after consuming the treatment beverage, the
second muscle biopsy was obtained (Figure 3.1). The second biopsy was taken ~1 cm
proximal to the first biopsy. Biopsy samples were quickly rinsed, blotted and divided into two
to three pieces, before being frozen in liquid nitrogen and stored at -80°C until later analysis.
Arterialized blood samples from a heated hand vein were collected at rest, immediately post-
exercise and every 15 min following beverage consumption for 2 h. Thereafter, blood
samples were obtained at regular intervals for the remainder of the infusion. Blood was
collected in EDTA-containing, lithium heparin-containing and serum separator tubes and
spun at 3500rpm for 15 min at 4°C. Aliquots of plasma and serum were the frozen at -80°C
until later analysis.
-180 2400 60 120 180-120 -60
* * * * ** * * ** * * *
Exercise
Time (min)
Biopsy
Blood
Primed-continuous L-[ring-¹³C6] phenylalanine infusion
Beverage
Figure 3.1 Schematic diagram of the experimental protocol.
Chapter 3. 106
Treatment beverages Immediately after the first muscle biopsy sample was obtained,
subjects ingested either 25.2g of carbohydrate (CHO) or 25.4g of carbohydrate plus 10.2g of
whey protein (C+P) dissolved in 250 mL of cold water (~11 ± 0 min post-exercise). A second
identical beverage was consumed 30 min after the first beverage was finished. This dose
regime provided a total carbohydrate and protein intake of 50.8g and 20.4g, respectively in
C+P and a total carbohydrate intake of 50.3g in CHO. Participants were encouraged to
consume the beverages within 2 min. Both CHO and C+P treatment beverages were matched
for flavour (orange and passion fruit) and appearance. Beverages were administered to
participants in a single-blinded manner, the order of which was randomized. Eight out of the
ten participants correctly identified the order of the treatments. The amino acid content of the
whey protein was (in percent content, wt:wt): Ala, 5.2; Arg, 2.2; Asp, 11.4; Cys, 2.3; Gln,
18.8; Gly, 1.5; His, 1.8; Ile, 6.7; Leu, 11; Lys, 10; Met, 2.3; Phe, 3.1; Pro, 5.7; Ser, 4.8; Thr,
7; Trp, 1.5; Tyr, 2.7; and Val, 6.1. A small amount of L-[ring-13
C6] phenylalanine tracer was
added to the C+P drink (6% of phenylalanine content) in order to minimize changes in blood
phenylalanine enrichment after drink ingestion.
3.3.5 Analyses
Blood analyses: Plasma glucose, plasma lactate and plasma urea concentrations were
analyzed using an ILAB automated analyzer (Instrumentation Laboratory, Cheshire, UK).
Serum insulin was analyzed using a commercially available ELISA kit (IBL International,
Hamburg, Germany), following the manufacturer’s instructions. Concentrations of
phenylalanine, leucine, threonine and 13
C6 phenylalanine tracer-to-tracee (t/T) enrichment
were determined by Gas Chromatography Mass Spectrometry (GCMS) (model 5973; Hewlett
Packard, Palo Alto, CA). On thawing, plasma samples were diluted 1:1 in acetic acid and
purified on cation-exchange columns (Dowex 50W-X8-200, Sigma-Aldrich Poole, UK). The
Chapter 3. 107
amino acids were then converted to their N-tert-butyldimethyl-silyl-N-
methyltrifluoracetamide (MTBSTFA) derivative. Plasma 13
C6 phenylalanine enrichment was
determined by monitoring at ions 234/240. Appropriate corrections were made for any
spectra that overlapped, contributing to the t/T ratio. Amino acid concentrations were
determined using an internal standard method (49, 51), based on the known volume of blood
and internal standard added. The internal standards used were U-[13
C9-15
N] phenylalanine
(50µmol/l), L-[2H3] leucine (120µmol/l) and L-[
2H4] threonine (182µmol/l) added in a ratio
of 100 µl/ml of blood. Leucine, threonine and phenylalanine concentrations were determined
by monitoring at ions 302/308, 404/409 and 336/346, respectively.
Muscle tissue analyses: Muscle samples were analyzed for enrichment of L-[ring-13
C6]
phenylalanine in the intracellular pool and bound myofibrillar and mitochondrial protein
fractions. Intracellular amino acids were liberated from ~10-20 mg of muscle. The tissue was
powdered under liquid nitrogen using a mortar and pestle and 500 µl of 0.2M perchloric acid
was added. The mixture was centrifuged at 10,000g for 10 min. The pH of the supernatant
was then adjusted to 5–7 with 2M KOH and treated with 20µL of urease for removal of urea.
The free amino acids from the intracellular pool were purified on cation-exchange columns
(described above). Intracellular amino acids were converted to their MTBSTFA derivative
and 13
C6 phenylalanine enrichment determined by monitoring at ions 234/240 (as described
above).
Mitochondrial and myofibrillar protein isolation was achieved using a protocol adapted
from Wilkinson et al. (54). Approximately 70-100mg of muscle tissue was homogenized in a
2mL Eppendorf with a Teflon pestle in 10µL·mg-1
of ice-cold homogenizing buffer (0.1 mM
KCl, 50 mM Tris, 5 mM MgCl, 1 mM EDTA, 10 mM β-glycerophosphate, 50 mM NaF,
1.5% BSA, pH 7.5). The homogenate was spun at 1,000g for 10 min at 4ºC. The supernatant
Chapter 3. 108
was transferred to another Eppendorf tube and spun at 10,000g for 10 min at 4ºC to pellet the
sarcoplasmic mitochondria (SM). The supernatant was then removed and discarded. The
pellet that remained from the original 1,000g spin was washed twice with homogenization
buffer. A glass Dounce homogenizer and tight fitting glass pestle were used to forcefully
homogenize the pellet in homogenization buffer to liberate intermyofibrillar mitochondria
(IM). The resulting mixture of myofibrillar proteins (MYO) and IM was spun at 1,000g for
10 min at 4ºC to pellet out the MYO. The supernatant was removed and spun at 10,000g for
10 min at 4º C to pellet the IM. The MYO, SM and IM pellets were washed twice with
homogenizing buffer containing no BSA. The MYO fraction was separated from any
collagen by dissolving in 0.3 M NaCl, removing the supernatant and precipitating the
proteins with 1.0 M PCA. All samples were washed once with 95% ethanol. In order to
determine 13
C6 phenylalanine enrichment in the mitochondrial protein fraction, IM and SM
fractions were combined as per Wilkinson et al. (54). Mitochondrial and myofibrillar
fractions were then hydrolysed overnight at 110 ºC in 0.05 M HCl/Dowex 50WX8-200
(Sigma Ltd, Poole, UK) and the constituent amino acids purified on cation-exchange columns
(Dowex 50W-X8-200, Sigma-Aldrich Poole, UK). The amino acids were then converted to
their N-acetyl-n-propyl ester derivative. Phenylalanine labelling was determined by gas-
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS, Delta-plus XL,
Thermofinnigan, Hemel Hempstead, UK) by monitoring at ions 336/342 for labelled and
unlabelled CO2. Unfortunately during processing 2 samples were lost for the mitochondrial
fractions, therefore the data represent n = 8. No myofibrillar fractions were lost during
processing (n = 10).
We assessed the purity of our protein fractions on muscle tissue collected during a pilot
study. Western blots revealed a greater abundance of myosin heavy chain protein content in
the myofibrillar fraction and a greater abundance of cytochrome c oxidase protein content in
Chapter 3. 109
the mitochondrial fraction. In addition, we measured citrate synthase (CS) activity in a mixed
muscle homogenate and each of the IM and SM mitochondrial fractions during method
development. We found that the CS activity was ~4-fold higher in the mitochondrial fractions
than the mixed homogenate. Data for these experiments is presented in Chapter 6 (sub-
section 6.1) of this thesis.
Western Blots: The remaining muscle tissue (25-40mg) was powdered on dry ice under
liquid nitrogen using a mortar and pestle. Approximately 20mg of powdered muscle was
homegenized in lysis buffer (50mM Tris pH 7.5; 250mM Sucrose; 1mM EDTA; 1mM
EGTA; 1% Triton X-100; 1mM NaVO4; 50mM NaF; 0.50% PIC), using a hand-held
homogenizer (PRO200, UK). Samples were shaken at 4ºC for 30 min (12,000rpm),
centrifuged for 5 min at 6,000g and the supernatant removed for protein determination.
Protein concentration was determined using the DC protein assay (Bio Rad, Hertfordshire,
UK). Equal aliquots of protein were boiled in Laemmli sample buffer (250mM Tris-HCl, pH
6.8; 2% SDS; 10% glycerol; 0.01% bromophenol blue; 5% β-mercaptoethanol) and separated
on SDS polyacrylamide gels (10 – 12.5%) for 1 h at 58mA. Following electrophoresis;
proteins were transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany)
at 100 V for 1 h. The membranes were incubated overnight at 4°C with the appropriate
primary antibody. The primary antibodies used were AMPKThr172
(Millipore 15-115),
mTORSer2448
(Cell signalling 2976), S6KThr389
(Cell signalling 9234), AktThr308
(Cell
signalling 4056), 4E-BP1Thr37
(Santa Cruz SC6025), eEF2Thr56
(Cell signalling 2332),
PRAS40Thr246
(Cell Signalling 2610) and p38 MAPKThr180
(Cell Signalling 9212). The
following morning the membrane was rinsed in wash buffer (TBS with 0.1% Tween-20)
three times for 5 min. The membrane was then incubated for 1 h at room temperature within
wash buffer containing the appropriate secondary antibody, either horseradish (HRP)-linked
Chapter 3. 110
anti-mouse IgG (New England Biolabs, 7072; 1:1,000) or anti-rabbit IgG (New England
Biolabs, 7074; 1:1,000). The membrane was then cleared in wash buffer three times for 5
min. Antibody binding was detected using enhanced chemiluminescence (Millipore,
Billerica, MA). Imaging and band quantification were carried out using a Chemi Genius
Bioimaging Gel Doc System (Syngene, Cambridge, UK). Intracellular signalling targets were
determined with n = 8 for CHO and C+P trials.
3.3.6 Calculations
The fractional synthetic rate (FSR) of mitochondrial and myofibrillar proteins were
calculated using the standard precursor-product method:
FSR (%·h-1
) = ∆Eb/Ep × 1/t × 100 (1)
Where ∆Eb is the change in bound 13
C6 phenylalanine enrichment between two biopsy
samples, Ep is the precursor enrichment and t is the time between muscle biopsies.
The true precursor enrichment would be the labelled phenylalanine-tRNA (1).
However, measurement of this enrichment requires large amounts of tissue, which cannot
typically be obtained from human volunteers. Thus, the intracellular (IC) free phenylalanine
enrichment is commonly used in studies (27, 38, 49), primarily because it is considered a
superior precursor surrogate. Unfortunately, due to technical difficulties, the IC enrichment
was available for only 5 participants. Thus, we chose to use the plasma precursor to estimate
the IC enrichment. A comparison of plasma and IC enrichments from the samples available
(n =5) revealed the IC enrichment to be 70 ± 2% of the plasma (range 67-77%). Furthermore,
a comprehensive examination of studies (for example; (2, 30, 46)) in which phenylalanine
Chapter 3. 111
tracers were used to determine FSR revealed the IC phenylalanine enrichment to be ~70% of
the plasma enrichment. We chose not to use the plasma enrichment, per se, as the precursor
for ease of comparison of the results to other previously published studies.
3.3.7 Statistical analysis
A within-subject repeated measures design was utilized for the current study. Exercise
variables, blood analytes and Western blot data were analyzed using a two-way ANOVA
with repeated measures (treatment x time) to determine differences between each treatment
beverage across time. Myofibrillar and mitochondrial FSR data were analyzed using one-
factor (treatment) repeated measures ANOVA. When a significant main effect or interaction
was identified, data were subsequently analyzed using a Bonferroni post hoc test. All
statistical tests were analyzed using statistical package for social sciences (SPSS) version
18.0 (Illinois, Chicago, U.S). Significance for all analyses was set at P < 0.05. All values are
presented as means ± standard error of the mean (SEM).
3.4 Results
3.4.1 Exercise variables
There was no between-trial difference in heart rate, cadence, V&O2 and RER measured at
25-30, 55-60 and 85-90 min of the steady state cycle (Table 3.1). Average HR over the 90
min cycle was 172 ± 3 and 172 ± 4 bpm for CHO and C+P, respectively. Average V&O2 over
90 min (51.3 ± 1.6 mL·kg-1·min
-1 for CHO and 51.6 ± 1.2 mL·kg
-1·min
-1 for C+P) and RER
over 90 min (0.88 ± 0.01 for CHO and C+P) were not different between treatments.
Metabolic data indicated participants were cycling at 77 ± 1% of V&O2max during CHO and
C+P trials. Average RPE over 90 min was similar for CHO and C+P (16 ± 4 for CHO and
C+P).
Chapter 3. 112
3.4.2 Blood analytes
Fasted blood glucose was 5.1 ± 0.3 mmol·L and 5.3 ± 0.3 mmol·L for CHO and C+P,
respectively and remained similar immediately post-exercise. Approximately 30 min after
consuming the first treatment beverage, plasma glucose concentration increased by ~32% and
~20% for CHO and C+P, respectively, with no significant difference between treatments.
Plasma glucose concentration returned to basal values by 1.5 h post-exercise for CHO and
C+P and was constant for the remainder of the infusion. Fasted serum insulin concentration
was 6.0 ± 0.8 and 5.5 ± 0.5 µU·ml-1
for CHO and C+P, respectively (Figure 3.2A). Following
drink ingestion, serum insulin concentration increased for both CHO and C+P, peaking at 30
min post-exercise (P < 0.001). Serum insulin increased to a greater extent for C+P (~285%)
compared with CHO (~60%; P < 0.001). Serum insulin returned to basal values by 1.5 h
post-exercise for CHO and C+P and remained constant until the end of the infusion.
Following exercise, plasma lactate concentration increased 150% and 175% compared with
resting values for CHO and C+P, respectively (P < 0.001), with no difference between
treatments. Lactate concentration returned to basal values by 3 h post-exercise for CHO and
C+P. Resting plasma urea concentration was similar for CHO and C+P and was stable
immediately post-exercise (Figure 3.2B). Following the ingestion of C+P, plasma urea
concentration increased by ~20% compared with resting values and remained elevated at 4 h
post-exercise (P < 0.05). Plasma urea concentration remained unchanged for CHO compared
with resting values. From 15 min to 4 h post-exercise, plasma urea concentration for C+P was
significantly greater than CHO (P < 0.05).
3.4.3 Plasma amino acid concentrations
Prior to and immediately post-exercise, plasma amino acid concentrations of
phenylalanine, leucine and threonine were similar for CHO and C+P (Figure 3.3). Following
Chapter 3. 113
the ingestion of C+P, plasma concentrations of phenylalanine, leucine and threonine
increased by 37, 130 and 58%, respectively (P < 0.001) and peaked at 1 h post-exercise (30
min after the second drink was ingested), after which, amino acid concentrations returned to
basal levels such that there were no differences between CHO and C+P at 4 h post-exercise.
Following the ingestion of CHO, plasma phenylalanine and threonine concentrations did not
change. Plasma leucine concentration was reduced following CHO ingestion (P = 0.04)
returning to pre-exercise levels by 2 h post-exercise.
3.4.4 Plasma and intracellular 13
C6 phenylalanine enrichment
Plasma 13
C6 phenylalanine enrichment increased from immediately pre- to post-
exercise but was stable across the time of tracer incorporation, immediately post-exercise to 4
h post-exercise for CHO and C+P (7.2 ± 0.03% t/T for both; Figure 4). These data
demonstrate that the additional tracer added to the C+P treatment beverage did not appear in
the circulation more rapidly than the amino acids from the protein and that all measurements
were made at isotopic equilibrium. Intracellular 13
C6 phenylalanine enrichment for an
available sub-set of participants (n = 5) was stable across the time of tracer incorporation for
CHO and C+P (4.8 ± 0.5 and 4.5 ± 0.7% t/T, respectively). Based on the relationship between
plasma and intracellular enrichments, the mean predicted intracellular enrichment for the
complete study cohort (n = 10) was 5.0 ± 0.1 and 5.1 ± 0.2% t/T for C+P and CHO,
respectively. Trial order did not influence the tracer enrichment in plasma (7.0 ± 0.3 and 7.4
± 0.2% t/T for trials 1 and 2, respectively; P = 0.2) or in the available intracellular samples
(4.5 ± 0.4 and 4.8 ± 0.3% t/T for trials 1 and 2, respectively; P = 0.2).
3.4.5 Post-exercise protein phosphorylation
Chapter 3. 114
Immediately post-exercise, mTORSer2448
phosphorylation was similar for CHO and
C+P. At 4 h post-exercise mTOR phosphorylation showed a tendency to increase for C+P (P
= 0.08), whereas mTOR phosphorylation tended to decrease for CHO (P = 0.07). At 4 h post-
exercise mTOR phosphorylation was greater for C+P compared with CHO (P = 0.02; Figure
3.5A). Immediately post-exercise, eEF2Thr56
phosphorylation was similar for CHO and C+P.
At 4 h post-exercise phosphorylation increased 1.4-fold for CHO compared with immediately
post-exercise (P = 0.02). Furthermore, eEF2 phosphorylation for CHO was greater than C+P
at 4 h post-exercise (P = 0.04; Figure 3.5B). Immediately post-exercise, p38 MAPKThr180
phosphorylation was similar for CHO and C+P. At 4 h post-exercise p38 MAPK
phosphorylation was unchanged for CHO and tended to increase for C+P (P = 0.08) with no
significant difference between treatments (Table 3.2). Immediately post-exercise,
p70S6KThr389
phosphorylation was similar for CHO and C+P. The phosphorylation of
p70S6K increased at 4 h for C+P compared with immediately post-exercise (P = 0.05) but
was not significantly different from CHO (Figure 3.5C). Immediately post-exercise, 4E-
BP1Thr37
phosphorylation was similar for CHO and C+P. At 4 h post-exercise 4E-BP1
phosphorylation showed a tendency to increase for CHO and C+P (P = 0.09) with no
difference between treatments (Table 3.2). There was no difference in the phosphorylation of
AMPKThr172
, AktThr308
and PRAS40Ser246
immediately post-exercise between CHO and C+P.
Furthermore, the phosphorylation of AMPK, Akt and PRAS40 remained unchanged at 4 h
post-exercise for CHO and C+P (Table 3.2).
3.4.6 Mitochondrial and myofibrillar FSR
Mitochondrial protein synthesis rates were similar for CHO and C+P (confidence
interval [CI]: 0.06 - 0.12 and 0.06 - 0.10 %·h
-1, respectively; Figure 3.6). Myofibrillar protein
synthesis rates were ~35% higher for C+P compared with CHO (CI: 0.07 - 0.11 and 0.05 -
Chapter 3. 115
0.07 %·h-1
, respectively; P = 0.025). Rates of mitochondrial and myofibrillar protein
synthesis were 30 ± 0.9% lower when the unadjusted plasma precursor was used in the
calculation of FSR. However, the statistical outcome was not different (data not presented).
CHO C+P
Ex time (min)
25-30 55-60 85-90 25-30 55-60 85-90
HR (bpm)
172 ± 5 172 ± 3 172 ± 1 174 ± 4 172 ± 1 171 ± 2
Cadence (rpm)
*84 ± 5 79 ± 5 80 ± 3 *84 ± 4 80 ± 3 79 ± 5
V&O2 (mL·kgˉ1·minˉ
1)
53.2 ± 2.0 50.7 ± 2.0 50.9 ± 2.0 52.6 ± 1.9 50.5 ± 1.9 50.9 ± 2.6
%V&O2max
80 ± 3 77 ± 2 76 ± 5 79 ± 2 76 ± 2 77 ± 3
RER
0.89 ± 0.01 0.87 ± 0.02 0.87 ± 0.02 0.91 ± 0.01 0.87 ± 0.02 0.87 ± 0.01
RPE 16 ± 1 17 ± 1 17 ± 1 16 ± 1 17 ± 1 18 ± 1
Table 3.1 Exercise variables
Values are the average recording over each 5 min phase and are presented as mean ± SEM. * indicates significant
difference at 25-30 min compared with other time phases (P < 0.05).
Chapter 3. 117
Figure 3.2 (A) Plasma urea and (B) serum insulin concentration in CHO and C+P trials. Means
with different subscripts are significantly different from each other (P < 0.05). *: significant
difference between CHO and C+P. Values are means ±SEM; n = 10.
Time (min)
-90 -60 -30 0 30 60 90 120 150 180 210 240
Seru
m insulin
concentr
ation (
µU
/mL)
0
5
10
15
20
25
30
35
40 C+P
CHO
a
a
b
c
a
aa
*
*
b
Exercise
b
aa a
aa
A
Time (min)
-90 -60 -30 0 30 60 90 120 150 180 210 240
Pla
sm
a u
rea
con
ce
ntr
atio
n (
mm
ol/L
)
0
3
4
5
6
7
CHO
C+P
b bb b
b b
Exercise
b
a
a
a a a aa a a
aa
*
B
Chapter 3. 118
Time (min)
-180 -120 -60 0 60 120 180 240
Pla
sm
a p
he
nyla
lanin
e c
on
ce
ntr
atio
n (
µm
ol/L)
0
40
50
60
70
80
90
100CHO
C+P
a
b
b
bcc c c
b
b
aa ab
ab
a
a cc
ac
aba a
*
Exercise
A
Time (min)
-180 -120 -60 0 60 120 180 240
Pla
sm
a le
ucin
e c
oncentr
ation (
µm
ol/L)
0
50
100
150
200
250
300
350CHO
C+P
a
a
a
b
b
c
c
c
b
b
ab
ab ab
a
bb b
b
c c cbc c
b
b b
*
Exercise
B
Chapter 3. 119
Time (min)
-180 -120 -60 0 60 120 180 240
Pla
sm
a t
hre
onin
e c
on
ce
ntr
atio
n (
µm
ol/L
)
0
50
100
150
200
250
300
350
CHO
C+P
a
ab abab
b b b b bb b
b b
a
aa a
a
b
b
b b
a a
aa
*
Exercise
Figure 3.3 Plasma concentrations of (A) phenylalanine, (B) leucine and (C) threonine. Means
with different subscripts are significantly different from each other. *: significant difference
between CHO and C+P (P < 0.05). Values are means ±SEM; n = 10.
Figure 3.4 Enrichment of 13
C6 phenylalanine in plasma. % t/T: percentage of tracer-to-tracee
ratio. Values are means ± SEM; n = 10.
Time post-exercise (min)
-90 -60 -30 0 30 60 90 120 150 180 210 240
Pla
sm
a L
-[ri
ng
-13C
6]
phe
nyla
lan
ine
en
rich
me
nt
(% t
/T)
0
4
5
6
7
8
9
10CHO
C+P
Exercise
C
Chapter 3. 120
Treatment CHO C+P
Time post-exercise (h) 0 - 4 0 – 4
AMPKThr172
0.95 ± 0.03
0.89 ± 0.08
AktThr308
0.92 ± 0.04
1.04 ± 0.04
4E-BP1Thr37
1.03 ± 0.02 1.04 ± 0.02
eEF2Thr56
1.4 ± 0.11*† 1.05 ± 0.12
mTORSer2448
0.91 ± 0.09 1.05 ± 0.05†
P38 MAPKThr180
1.15 ± 0.21 1.28 ± 0.08
p70S6KThr389
1.06 ± 0.04 1.13 ± 0.05*
PRAS40Ser246
0.97 ± 0.08 1.03 ± 0.08
Values are mean fold-change ± SEM over 4 hours post-exercise. * indicates significant increase
in phosphorylation at 4 h compared with 0 h post-exercise (P < 0.05). † indicates significant
difference between CHO and C+P at 4 h post-exercise (P < 0.05); n = 8
Table 3.2 Post-exercise protein phosphorylation
Chapter 3. 121
C+P CHO
p -
mT
OR
Ser2
448 (
Arb
itra
ry u
nits)
0.0
0.8
1.0
1.2
1.4
1.6
1.8
2.00h Post-ex
4h Post-ex
†
A
C+P CHO
p -
eE
F2 T
hr5
6 (
Arb
itra
ry u
nits)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0h Post-ex
4h Post-ex *
†
B
Chapter 3. 122
C+P CHO
p -
p7
0 S
6K
Thr3
89 (
Arb
itra
ry u
nits)
0.0
0.6
0.8
1.0
1.2
1.4
1.60h Post-ex
4h Post-ex *
C
Figure 3.5 Post-exercise protein phosphorylation of (A) mTORSer2448
, (B) eEF2Thr56
and (C)
p70S6KThr389
*: significant increase in phosphorylation at 4 h compared with 0 h post-exercise
(P < 0.05) †: significant difference between CHO and C+P at 4 h post-exercise (P < 0.05).
Values are means ± SEM; n = 8.
Chapter 3. 123
3.5 Discussion
The present study findings expand on those of previous investigations (27, 34) to show
that the ingestion of protein with carbohydrate (C+P) following 90 min of cycling by well-
trained individuals, stimulates an increase in myofibrillar muscle protein synthesis (MPS)
compared with carbohydrate alone (CHO). Interestingly, C+P did not increase mitochondrial
MPS compared with CHO. The mechanism facilitating the adaptive response of myofibrillar
MPS seems to involve the phosphorylation of mTOR and p70S6K, which in turn, may
increase translation initiation and release the inhibition of eEF2 on elongation.
It has been well documented that EE increases whole-body (9) and mixed muscle
protein synthesis (10, 18, 19, 50). Further, the synthesis of different protein sub-fractions
Myofibrillar Mitochondrial
FS
R (
%·h
-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12CHO
C+P*
Figure 3.6 Myofibrillar (n = 10) and mitochondrial (n = 8) fractional synthetic rate. *:
significant difference between CHO and C+P (P < 0.05). Values are means ±SEM.
Chapter 3. 124
increases in response to EE (37, 54). Changes in MPS following EE are thought to be
relevant for tissue repair and remodelling and not skeletal muscle hypertrophy. Indeed, it was
recently shown that EE stimulates an increase in the synthesis of mitochondrial proteins, with
no significant rise in myofibrillar MPS (54). The same authors demonstrate that the response
of mitochondrial and myofibrillar MPS is reversed following an acute bout of resistance
exercise (RE) (54). Post-RE protein ingestion also augments myofibrillar MPS (26). Given
evidence that the pattern of response of mitochondrial and myofibrillar proteins to the
provision of amino acids is similar under resting conditions (4, 5), it has been postulated that
the increase in mixed MPS observed when protein is ingested post-EE (27, 34), could be due,
in part, to an increase in the synthesis of mitochondrial proteins. However, our findings
seemingly contradict this hypothesis and indicate that myofibrillar proteins are preferentially
synthesized when protein is ingested following EE. Recently Harber et al. (19) showed no
response of mixed MPS to post-EE protein ingestion following a 60 min cycle. These authors
(19) measured mixed MPS at 2-6 hours post-EE, starting at 1 hour after feeding. Thus, the
peak anabolic response to exercise and feeding (32, 55) may have occurred earlier than was
measured, concealing any potential difference. Based on the data of Harber et al. (19), we
suggest that the measure of mixed MPS may not be sensitive enough to reflect changes in
myofibrillar and mitochondrial MPS.
The rise in myofibrillar MPS following a single bout of RE with protein ingestion (55),
is predictive of gains in lean muscle mass when the training and feeding stimulus is repeated
over an extended time-period (20). Thus, one interpretation of our data is that repeated post-
EE protein ingestion may enhance muscle hypertrophy in endurance athletes, which,
depending on the competitive goals of the athlete, may, or may not be a desirable adaptation.
Gains in lean mass may result in a decreased power-to-mass ratio that accompanies an
Chapter 3. 125
increase in overall body weight. Indeed, evidence indicates that the primary goal of strength
training for endurance populations is neuromuscular adaptations, rather than hypertrophy, to
enhance performance (40). However, we hypothesize that the increase in power output that
accompanies gains in lean mass may offset the gains in overall body weight, resulting in no
decrement in performance. In support of this thesis, Hickson et al. (23) demonstrated that
gains in thigh mass and thus, total body weight following strength training, extend short-term
endurance capacity. Thus, manipulation of the power-to-mass ratio in competitive cyclists
would likely have a profound impact on overall performance depending on the requirements
of the particular event (11, 31). At this time, the implications of muscle hypertrophy for
endurance performance are less than clear, as is the notion that post-EE protein ingestion
promotes muscle hypertrophy. Clearly, these topics warrant further investigation.
Alternatively, the increase in myofibrillar MPS we observed following exercise may
not lead to an increase in lean mass. Where the magnitude of myofibrillar muscle protein
breakdown (MPB) equals or exceeds that of myofibrillar MPS, structural proteins would not
accumulate to increase lean mass. Direct measurement of muscle protein breakdown with
endurance exercise is rare and the results somewhat equivocal (12, 15, 44). Using venous
blood sampling, other investigators have demonstrated that whole-body protein breakdown is
transiently elevated after moderate intensity walking exercise, compared with rest (44), but
others report no change following exercise (50). These studies investigated the response to
moderate to low intensity exercise, rather than the high intensity exercise performed by our
subjects. However, studies of endurance exercise have reported an increase in indirect
markers of MPB including 3-methyl-histidine excretion (10) and net amino acid efflux from
the leg (3, 52) during exercise. Thus, elevated myofibrillar MPS may also represent an
increase in protein turnover in response to an increase in the degradation of myofibrillar
Chapter 3. 126
proteins during and following intense exercise. Combining these data, it is possible that
increased myofibrillar MPS we observed when protein is ingested following intense EE,
serves to counteract the fasted state losses that occurred during exercise and thus, represents
an increase in myofibrillar protein turnover. Increased protein turnover may be important for
the remodelling of contractile proteins. However, this hypothesis is not supported by a recent
study by Harber et al. (19) that demonstrated that protein ingestion following an intense cycle
attenuates the expression of proteolytic factors such as MuRF-1 and calpain-2. However, the
relationship between the expression of these genes and protein degradation is uncertain (17).
Further studies are required to determine the influence of post-EE protein ingestion on
myofibrillar protein degradation and longitudinal intervention studies are needed to determine
the relevance of increased protein turnover to the adaptive response to EE and performance.
The addition of protein to ingested carbohydrate did not increase the synthesis of
mitochondrial proteins in the first 4 hours following exercise. Thus, the acute translational
response to EE (Wilkinson et al., 2008) may not be sensitive to protein ingestion. However,
the long-term response of mitochondria to EE is transcriptionally mediated and occurs later
than four hours post-EE (16, 21, 43), reaching a zenith between 10-24 hours post-EE (33, 37).
Combining these data with recent evidence that mixed MPS remains elevated above rest at 24
h post-EE (18), it is conceivable that the major response of mitochondrial MPS to post-EE
protein ingestion may have occurred later than the time frame in which we measured.
Another possibility is that the training status of the subjects and the exercise protocol
utilized in the present study may explain the lack of change in mitochondrial MPS. Indeed,
compared with pre-training values, Wilkinson et al. (54) found that the response of
mitochondrial MPS to acute EE was blunted in legs that had undergone 10 weeks of either
Chapter 3. 127
EE or RE training. Thus, it is possible that the synthetic response of mitochondrial proteins in
well-trained individuals reaches a ‘ceiling’; the point beyond which, further contractile and/or
nutrient stimuli fail to augment the synthetic response. Whether the pattern of myofibrillar
and mitochondrial MPS we found is the same in untrained individuals remains to be
elucidated.
EE can activate proteins that regulate translation initiation (7, 36, 54) (i.e. mTOR) and
elongation (36) (i.e. eEF2). The transient increase in mTOR signalling following EE (7, 36),
returns to basal levels by 3-4 hours post-EE (54, 56). Fujita et al. (14) and others have shown
that ingesting protein and/or amino acids at rest increases MPS via mTOR-p70S6K
signalling. To our knowledge, the only study in humans to investigate the combined effect of
EE and protein ingestion on signalling protein phosphorylation was conducted by Ivy et al.
(28). In this study, trained cyclists ingested C+P over 45 minutes after a 1 hour variable
intensity cycle. The authors demonstrated that the C+P group showed higher mTOR and rpS6
phosphorylation than a non-energetic placebo. However, due to the study design, it was
unclear whether these effects were due to greater amino acid availability, elevated plasma
insulin concentration, or a combination of the two following C+P ingestion. The same group
had earlier conducted a similar, more rigorous experiment, in which rats were fed
immediately after an exhaustive swim (39). The authors demonstrated that ingesting CHO,
C+P or protein-only, transiently increased the phosphorylation of mTOR, 4E-BP1, rpS6 and
p70S6K, with the phosphorylation of rpS6 and 4E-BP1 sustained by C+P ingestion (39). Our
data show that protein ingestion following EE sustained the phosphorylation of mTOR, such
that by 4 hours post-exercise the difference between C+P and CHO was significant. Further,
p70S6K phosphorylation increased by 4 hours post-exercise for C+P, compared with
Chapter 3. 128
immediately post-exercise. Thus, since mTOR activation is associated with increased
initiation, this suggests that C+P prolongs the effect of EE on protein synthesis.
In addition to initiation, it is now recognized that translation elongation is regulated by
exercise and nutrient provision. Reduced phosphorylation of eEF2 due to eEF2 kinase
inhibition results in better translocation of the ribosome along the mRNA, thereby
contributing to faster elongation and greater MPS (8). Mascher et al. (36) showed that eEF2
phosphorylation was blunted following EE, reaching a nadir at 60 min and remaining
suppressed at 180 min post-EE. Concomitant with an increase in MPS, Fujita et al. (14) noted
that essential amino acid-carbohydrate co-ingestion suppressed the phosphorylation of eEF2.
Thus, eEF2 phosphorylation is sensitive to EE and protein ingestion. In this regard, we show
that eEF2 phosphorylation increased by ~40% at 4 h post-EE in the CHO group, whereas
there was no change with protein ingestion. Our findings are further supported by human (14)
and cell culture (53) studies showing that mTOR regulation of p70S6K is inversely related to
eEF2 phosphorylation. Taken together, these data suggest that the sustained phosphorylation
of p70S6K, suppressed the phosphorylation of eEF2 following C+P ingestion, thereby
increasing the rate of elongation. Together with the increase in initiation due to prolonged
mTOR activation, this would lead to a rise in myofibrillar MPS.
In conclusion, we have shown that when protein is added to carbohydrate intervention
following cycling exercise, myofibrillar proteins are preferentially synthesized. However,
from our acute study it is not possible to determine whether frequent post-EE protein
ingestion promotes muscle hypertrophy over time. Alternatively, the rise in myofibrillar MPS
with protein ingestion, may serve to counteract a fasted-state rise in myofibrillar protein
breakdown during EE. Consequently, protein ingestion may increase the rate of myofibrillar
Chapter 3. 129
protein turnover. The importance of an increase in myofibrillar protein turnover for
endurance-trained individuals remains to be elucidated, but may be relevant for the
maintenance and structural integrity of contractile proteins. Thus, we posit that post-EE
protein nutrition could have important implications on the adaptive response to endurance
exercise and, potentially, the recovery of muscle function. Finally, the synthetic response of
different protein fractions to endurance exercise and protein ingestion may be dependent on
the individual training status, intensity and duration of the exercise bout, as well as the timing
and quantity of post-EE nutrient strategies. Future studies should seek to address these issues
in greater depth.
Author contributions
L.B., O.C.W., S.R.J., K.B. and K.D.T. contributed to the conception and design of the
experiment. L.B., A.P., O.C.W., S.R.J. and K.D.T. contributed to the collection of the data.
L.B., A.P., O.C.W., S.R.J., A.S., K.S., M.J.R., K.B. and K.D.T. contributed to the analysis
and interpretation of the data. L.B., A.P., K.S., M.J.R., K.B. and K.D.T. contributed to
drafting or revising the content of the manuscript.
The authors declare no conflict of interest.
Acknowledgements
We would like to thank Dr Daniel Moore for his cooperation during method
development and Ms Lorna Webb for assistance during data collection. We would also like to
extend our appreciation to the participants for their time and effort. This study was funded by
a research grant from GlaxoSmithKline Nutritional Healthcare to K.D.T.
Chapter 3. 130
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56. Witard OC, Tieland M, Beelen M, Tipton KD, van Loon LJ, and Koopman R.
Resistance exercise increases postprandial muscle protein synthesis in humans. Med Sci
Sports Exerc 41: 144-154, 2009.
Chapter 4 135
CHAPTER 4
BENEFICIAL EFFECTS OF RESISTANCE EXERCISE ON GLYCEMIC CONTROL
ARE NOT FURTHER IMPROVED BY PROTEIN INGESTION
Leigh Breen1, Andrew Philp
2, Christopher. S. Shaw
1, Asker. E. Jeukendrup
1, Keith Baar
2, and
Kevin. D. Tipton1, 3
1School of Sport and Exercise Sciences, University of Birmingham, Birmingham, United
Kingdom ,2Functional Molecular Biology Lab, Neurobiology, Physiology and Behaviour,
University of California Davis, US. 3
Department of Sports Studies, University of Stirling,
Stirling, UK.
Running title: Influence of protein ingestion on post-exercise glucose regulation.
Address for Correspondence:
Kevin. D. Tipton, Ph.D.
The University of Stirling
Department of Sports Studies
Stirling, UK.
FK9 4LA
Email: [email protected]
Chapter 4
136
4.1 Abstract
Purpose: To investigate the mechanisms underpinning modifications in glucose
homeostasis and insulin sensitivity at 24 h after a bout of resistance exercise (RE) with or
without protein ingestion. Methods: Twenty-four healthy males were assigned to a control
(CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). EX
and EX+PRO completed 8 x 10 repetitions of leg press and leg extension exercise at 75%
1RM. Muscle biopsy and blood samples were obtained for all groups at rest and immediately
post-RE for EX and EX+PRO. At 24 h post-RE (or post-resting biopsy for CON), a third
muscle biopsy was obtained. Participants then underwent an oral glucose tolerance test
(OGTT) containing 2g of [U-13
C] glucose. Thereafter, blood samples were obtained every 10
min for 2 h. Glucose kinetics were assessed with an infusion of 6, 6-[2H2] glucose during the
OGTT. EX+PRO ingested an additional 25g of intact whey protein with the OGTT. A final
biopsy sample was obtained at the end of the OGTT. Biopsies were analyzed for insulin-
stimulated signalling phosphorylation and muscle glycogen content. Results: Basal, fasting
plasma glucose and insulin were similar for all groups and were not different immediately
post- and 24 h post-RE. Immediately post-RE, muscle glycogen was 26 ± 8 and 19 ± 6%
lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for
EX and EX+PRO (75.1 ± 2.7 and 75.3 ± 2.8 mU·mL-1
: 120 min, respectively) compared with
CON (90.6 ± 4.1 mU·mL-1
: 120 min; P < 0.05). Plasma insulin response was 13 ± 2 and 21 ±
4% lower for EX and CON, respectively, compared with EX+PRO (P < 0.05). Post-prandial
insulin sensitivity was greater for EX and EX+PRO compared with CON (P < 0.05). Glucose
disappearance from the circulation was ~12% greater in EX and EX+PRO compared with
CON (P < 0.05). Basal and insulin-stimulated PAS-AS160 phosphorylation was greater for
EX and EX+PRO (P < 0.05). Conclusions: Prior RE improves glycemic control and insulin
sensitivity through an increase in the rate at which glucose is disposed from the circulation.
Chapter 4
137
However, co-ingesting protein during a high-glucose load does not augment this response at
24 h post-exercise.
Keywords: Type II diabetes, insulin resistance, physical activity, nutrition, skeletal muscle.
Abbreviations: GLUT4; glucose transporter protein, RE; resistance exercise, AMPK; AMP-
activated protein kinase, Akt; protein kinase B, AS160; Akt-substrate of 160-kDa, TBC1D1;
TBC1 domain family member 1, p70S6K; ribosomal protein S6 kinase, mTOR; mammalian
target of rapamycin.
Chapter 4
138
4.2 Introduction
Metabolic complications, such as insulin resistance and Type II diabetes, represent a
major individual and public health burden (25) and are associated with the presence of
obesity and physical inactivity (13, 22, 41). Suppressed pancreatic β cell insulin production
(7) and impaired glucose uptake in skeletal muscle (8, 70), are major contributors to
hyperglycemia, which eventually leads to Type II diabetes. Insulin stimulates the uptake of
glucose from the circulation into many body tissues, of which, skeletal muscle accounts for
~75-80% (9, 56). Insulin-stimulated glucose uptake in skeletal muscle occurs via insulin-
dependent signalling that promotes glucose transporter (GLUT4) translocation to the cell
membrane. Even though pharmacologic approaches are available to manage Type II diabetes,
there is growing interest in lifestyle interventions, such as physical activity and dietary
modification, which together can reduce the incidence of diabetes to a greater extent than
pharmacological agents (61).
Skeletal muscle contraction can effectively enhance glucose uptake independent of
insulin. Importantly, these properties are preserved in individuals with Type II diabetes (19,
28). A number of studies have demonstrated that aerobic exercise improves glycemic control
and insulin sensitivity (10, 11, 40, 50). More recently, evidence has emerged to suggest that a
single bout of resistance exercise (RE) improves whole-body insulin sensitivity (15, 30) and
glycemic control (14) between 12 and 24 h post-RE. However, not all studies demonstrate a
beneficial effect of RE on glucose metabolism (5, 33). The discrepant findings between
studies may be due to methodological differences, including, the total exercise volume, the
muscle groups exercised, the amount of time between the exercise and testing, and individual
training status.
Chapter 4
139
The glucose transporter GLUT4 plays an important role in skeletal muscle glucose
homeostasis by regulating glucose uptake (23). Insulin regulates GLUT4 translocation from
an intracellular location to the cell surface via a well-described pathway involving protein
kinase B (Akt) and the recently discovered GTPase, Akt substrate of 160kDa (AS160) (21).
Although enhanced glucose uptake with acute RE is thought to occur via mechanisms that
bypass proximal insulin-signalling intermediates, such as Akt (3, 6, 29), AS160 appears to act
as a point of convergence for insulin- and contraction-stimulated glucose transport in skeletal
muscle (21, 32, 58). Recently, basal and insulin-stimulated AS160 phosphorylation was
shown to increase several hours after endurance and resistance exercise in humans (12, 21)
and up to 27 h post-exercise in animals (16, 60). However, the sustained response of AS160
has not been investigated in humans. Thus, the molecular and physiological mechanisms by
which RE improves glucose metabolism in the 24 h after exercise have yet to be elucidated.
In addition to RE, dietary modifications that acutely raise endogenous insulin secretion,
represent a clinically relevant strategy to improve blood glucose homeostasis in Type II
diabetes. A number of recent studies suggest that co-ingesting protein and/or amino acids
with carbohydrate induces a greater insulin release than the ingestion of either macronutrient
alone (46, 49, 62). Indeed, the rise in plasma insulin with carbohydrate plus protein ingestion
blunts the prevailing glucose response in Type II diabetics (34, 36), primarily due to an
increase in the rate of glucose disposal from the circulation (36). Thus, in persons with Type
II diabetes, co-ingesting protein with each main meal may be an effective strategy to acutely
lower postprandial glucose excursions (44, 46). The combined effect of protein ingestion and
RE has largely been considered in the context of muscle hypertrophy (42, 59). However, the
hypothesis that post-RE protein ingestion acutely enhances glucose uptake has not been
investigated in humans.
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140
In the present study we used dual isotopic tracers during an oral glucose load to first
determine the impact of an acute bout of RE on post-prandial glucose kinetics 24 h post-
exercise. Second, we aimed to determine whether co-ingesting protein with the glucose load
would further augment the glucose-lowering effect of the RE bout. Finally, we obtained
muscle biopsy samples to examine the mechanistic basis of the effect of RE on glycemic
control.
4.3 Methods
4.3.1 Participants
Twenty-four untrained, recreationally active, healthy males were recruited through
advertisements to participate in the study. Individuals who were engaged in regular structured
resistance or endurance training, defined as ≥ 2 training sessions per week of 60 mins or
more, were ineligible to participate. All testing visits were completed within a 3-week period.
The purpose and methodology of the study were clearly explained to the participants. All
participants gave their informed consent prior to taking part in the study and were deemed
healthy based on their response to a general health questionnaire. The experimental protocol
was approved by the NHS Birmingham East, North & Solihull Research Ethics Committee
(Rec No: 09/H1206/102).
4.3.2 Study design
In a randomized, parallel designed study, participants were assigned to either a non-
exercise control (CON; n = 8), exercise only (EX; n = 8) or exercise plus protein (EX+PRO; n
= 8) condition. Participant characteristics are presented in Table 4.1. Following a preliminary
assessment of maximal lower-limb strength, participants reported to the laboratory in a fasted
Chapter 4
141
state, on two consecutive mornings. On the first morning, a resting muscle biopsy sample was
obtained, thereafter EX and EX+PRO performed an intense lower-limb workout. A second
biopsy was obtained immediately post-exercise for EX and EX+PRO only. Twenty-four hours
later another muscle biopsy was obtained, after which, participants completed an oral glucose
tolerance test (OGTT). Participants assigned to EX+PRO co-ingested protein with the OGTT
to determine whether the addition of protein augmented the impact of resistance exercise on
glucose metabolism. During OGTT, dual isotopic glucose tracers were utilized and frequent
blood samples obtained over 2 h to determine glucose kinetics and insulin sensitivity. A final
muscle biopsy was obtained at the end of the 2 h OGTT to examine the phosphorylation of
contraction- and insulin-mediated intracellular protein phosphorylation.
Parameter
CON (n = 8)
EX (n = 8)
EX+PRO (n = 8)
Age (y)
22 ± 3
20 ± 3
22 ± 6
Weight (kg)
77.0 ± 6.3 79.7 ± 14.5 75.6 ± 13.1
BMI (kg·m-2
)
24 ± 2.3 25.1 ± 3.9 23.5 ± 4.8
LP 1RM (kg) 200 ± 44 212 ± 50 206 ± 60
LP 1RM (kg·BM-1
)
5.7 ± 0.8 5.9 ± 1.8 6.0 ± 1.3
LE 1RM (kg)
111 ± 22 121 ± 21 115 ± 29
LE 1RM (kg·BM-1
)
3.5 ± 1.2 3.7 ± 0.7 3.4 ± 0.6
Exercise volume (kg) ¯ 18,314 ± 2154 18,277 ± 2406
CON: resting control group, EX: exercise only group, EX+PRO: exercise plus protein group
BMI; body mass index, 1RM; one-repetition maximum, LP: leg press, LE; leg extension.
Exercise volume is defined as number of repetitions x number of sets x weight lifted. Values
are presented as means ± SD.
Table 4.1 Characteristics of participants in each group
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142
4.3.3 Preliminary testing
Body Mass: A digital scale was used to determine body mass to the nearest 0.1kg.
Participant weight and height were recorded in exercise clothing without shoes on. This was
repeated prior to each of the two testing visits to ensure body mass remained stable
throughout testing.
Maximal Strength: Bilateral 1 Repetition Maximum (1RM) was determined for leg
press and leg extension exercises (Cybex VR/3). After warming up at a self selected
resistance, the load was set at a level designed to allow the subject to perform at least two, but
less than ten repetitions before failure. This estimation protocol of 1RM was designed to
minimise the number of attempts necessary to determine 1RM (39). After each successful lift
the load was increased by 2.5-5 kg until failure to complete two repetitions. Between each
successive attempt a 2 min rest period was allowed. A repetition was considered valid if the
subject used proper form and was able to complete the entire lift in a controlled manner
without assistance.
Diet and Physical Activity Control: Participant diet was standardized for the entire 48 h
testing period. During preliminary testing participants completed a 3-day food diary,
representative of their average week (two week days and one weekend day). A questionnaire
of food preferences was also completed by participants. Using an on-line diet planner (Weight
Loss Resources) the total energy and macronutrient content of each of the 3-days was
estimated. Food parcels were provided to each participant with a total energy and
macronutrient intake equivalent to their average habitual intake. Thus, participant diet was not
manipulated during the study. Participants were instructed to consume only the food provided
for them over the two-day testing period (i.e. 24 h prior to Day 1 and during Day 1). There
Chapter 4
143
was no difference in total energy intake or macronutrient composition of the food parcels for
CON, EX and EX+PRO (Table 4.2).
Physical activity control: Participants were instructed to maintain normal habitual
activities of daily living but to refrain from any strenuous activity for 48 h prior to reporting to
the laboratory on Day 1. After completion of testing on Day 1, participants were also
instructed to refrain from strenuous activity prior to returning the following morning on Day
2.
4.3.4 Treatment trials
Day 1 - Exercise/Control Trial: Participants reported to the Human Performance
Laboratory between 0600 and 0700 h in a fasted state, 7-14 days after preliminary strength
tests. After resting in a supine position for 30 min a cannula was inserted into an antecubital
forearm vein and a resting blood sample obtained for analysis of background isotopic
enrichment. Thereafter, the lateral portion of one thigh was prepared under local anaesthetic
(1% Lidocaine) and a 5-mm Bergstrom biopsy needle was used to extract a resting muscle
biopsy sample from the vastus lateralis muscle. After the resting biopsy was obtained the
biopsied leg was bandaged and EX and EX+PRO were instructed to complete a bout of
intense lower-limb resistance exercise, whereas CON were permitted to consume the
standardized breakfast and leave the laboratory. For EX and EX+PRO a second biopsy
sample was obtained post-exercise (6 ± 1 min) 1 cm distal from the resting biopsy. For EX
and EX+PRO both biopsy incisions were made at rest to allow the post-exercise biopsy
sample to be obtained as quickly as possible. The order of biopsied leg was counterbalanced
between groups. Biopsy samples were blotted and freed of any visible fat and connective
Chapter 4
144
tissue, frozen in liquid nitrogen (within ~60 s of being taken from the muscle) and stored at -
80°C until further analysis.
The resistance exercise bout consisted of a standardized warm-up on a leg-press
machine (12 x 50% 1RM + 10 x 60% 1RM + 8 x 70% 1RM + 2 x 75% 1RM) followed by 8
sets of 10 bilateral repetitions at 75% 1RM. Participants then completed 8 sets of 10 bilateral
repetitions on a leg-extension machine at 75% 1RM. In the event that a participant failed to
complete 10 repetitions in a set the weight was decreased by 2.5-5kg for the following set.
Failure was defined as the point at which the exercise could not be completed or technique
failed. Participants were instructed on proper lifting cadence using a metronome set to 50
beats min−1
, which corresponded to 1 s concentric muscle action, 0 s pause and a 1 s eccentric
muscle action. Strong verbal encouragement was given throughout exercise. Between-set rest
intervals of 2 min were given and participants completed the exercise bout in 45 ± 3 min.
Participants were permitted to consume water ad libitum throughout Day 1.
Day 2 - Infusion Trial: Participants returned to the laboratory the following morning
between 0600 and 0700 h in a fasted state. A schematic diagram of the study protocol is
presented in Figure 4.1. A cannula was inserted into the antecubital vein of one arm for the
infusion of a stable isotopic tracer. A second cannula was inserted into a hand vein of the
opposite arm and a resting venous blood sample obtained. At a time-point corresponding to
~23 h post-exercise for EX and EX+PRO a primed infusion of 6, 6-[2H2] glucose (Cambridge
Isotope Laboratories, MA, USA) was initiated (prime: 13.5 µmol.kg-1
; infusion: 0.35 µmol.kg-
1.min
-1) and continued for ~180 min. For CON, the infusion was initiated at a time-point
corresponding to ~23 h after the resting biopsy on Day 1. Approximately 60 min into the
infusion (~24 h post-exercise or resting biopsy) a muscle biopsy sample was obtained from
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145
the vastus lateralis of the opposite leg to that sampled the previous day. Immediately after the
muscle biopsy was obtained CON and EX consumed an oral glucose load (oral glucose
tolerance test; OGTT) described below. EX+PRO consumed the same glucose load plus
additional protein. The time at which the beverage was completely consumed was considered
t = 0, thereafter participants rested in the supine position and venous blood samples were
obtained every 10 min until t = 120. A final muscle biopsy sample was obtained at t = 120
from a separate incision (~26 h post-exercise or resting biopsy). Water intake was restricted
during Day 2 to ensure participants consumed only the treatment beverage.
4.3.5 Treatment beverages
Sixty minutes into the infusion on Day 2, participants ingested 73g of glucose (80.3g
dextrose monohydrate when corrected for water content) plus an additional 2g of [U-13
C]
glucose (99%, Cambridge Isotope Laboratories, MA, USA) to determine the contribution of
exogenous glucose production to the total rate of appearance of glucose. Thus, participants
ingested a total of 75g of glucose. Participants assigned to EX+PRO ingested an additional
25g of whey protein with the 75g glucose load. Glucose was provided in the form of dextrose
monohydrate obtained from Roquette™ (Lestrem, France) and intact whey protein (Volactive
ultrawhey 90) was a generous gift from Volac™ (Royston, UK). The amino acid content of
the protein was (in percent content, wt:wt): Ala, 5; Arg, 2.1; Asp, 11; Cys, 2.2; Gln, 18.1;
Gly, 1.4; His, 1.7; Ile, 6.4; Leu, 10.6; Lys, 9.6; Met, 2.2; Phe, 3; Pro, 5.5; Ser, 4.6; Thr, 6.7;
Trp, 1.4; Tyr, 2.6; and Val, 5.9. Both treatments were consumed in water in a total volume of
300mL. Treatments were not matched for flavour or appearance due to the parallel study
design. Participants were instructed to consume the treatment beverage within 2 min.
Chapter 4
146
4.3.6 Analyses
Blood analytes: Blood was collected in EDTA-containing tubes and spun at 3,500rpm
for 15 min at 4°C. Aliquots of plasma were the frozen at -80°C until later analysis. Plasma
glucose concentration was analyzed using an ILAB automated analyzer (Instrumentation
Laboratory, Cheshire, UK). Plasma insulin concentration was analyzed using a commercially
available ELISA kit (IBL International, Hamburg, Germany) following the manufacturer’s
instructions. The enrichment of [13
C] and [2H2] glucose in plasma was determined by gas
chromatography-mass spectrometry (model 5973; Hewlett Packard, Palo Alto, CA).
Derivatization was carried out with butane boronic acid in pyridine and acetic anhydride. The
glucose derivative was quantified by selected ion monitoring at mass-to-charge ratios (m/z)
-1 0 21
Primed-continuous [6,6-2H2] glucose infusion
Time (h) -24-25
* ** *** **
Exercise
Biopsy
Blood
Beverage
* * * *** * * *
-1 0 21
Primed-continuous [6,6-2H2] glucose infusion
Time (h) -24-25
Rest
Biopsy
Blood
Beverage
** *** ** * * * *** * * *
Figure 4.1 Schematic diagram of the study protocol. (A) Indicates protocol for exercise only
and exercise plus protein participants. (B) Indicates protocol for non-exercised control
participants.
(A)
(B)
Chapter 4
147
297, 299 and 303 for [12
C]-, [2H2]- and [U-
13C] glucose, respectively. Two sets of enriched
standards were measured containing known amounts of [2H2]- and [U-
13C] glucose. By
establishing the relationship between the enrichment of the glucose standards, the enrichment
in plasma samples was determined.
Western blots: Muscle biopsy samples (~40mg) were powdered on dry ice under liquid
nitrogen using a mortar and pestle. Approximately 25mg of powdered muscle was
homegenized in lysis buffer (50mM Tris pH 7.5; 250mM Sucrose; 1mM EDTA; 1mM
EGTA; 1% Triton X-100; 1mM NaVO4; 50mM NaF; 0.50% PIC) using a hand-held
homogenizer (PRO200, UK). Samples were shaken at 4ºC for 30 min (12,000rpm),
centrifuged for 5 min at 6,000g and the supernatant removed for protein determination.
Protein concentration was determined using the DC protein assay (Bio Rad, Hertfordshire,
UK). Equal aliquots of protein were boiled in Laemmli sample buffer (250mM Tris-HCl, pH
6.8; 2% SDS; 10% glycerol; 0.01% bromophenol blue; 5% β-mercaptoethanol) and separated
on SDS polyacrylamide gels (10-12.5%) for 1 h at 58mA. Following electrophoresis; proteins
were transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) at 100 V
for 1 h. The membranes were incubated overnight at 4°C with the appropriate primary
antibody. The primary antibodies used were total Akt, Aktser473
(Cell signalling 3787), total
AS160 and phospho AS160 were a generous gift from Prof. Grahame Hardie, University of
Dundee, the PAS-AS160 antibody was generated by the division of signal transduction
therapy (DSTT), University of Dundee. Total p70S6K and phospho p70S6KThr389
was from
Santa Cruz (11759/7984R). The following morning the membrane was rinsed in wash buffer
(TBS with 0.1% Tween-20) three times for 5 min. The membrane was then incubated for 1 h
at room temperature within wash buffer containing the appropriate secondary antibody, either
horseradish (HRP)-linked anti-mouse IgG (New England Biolabs, 7072; 1:1,000) or anti-
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148
rabbit IgG (New England Biolabs, 7074; 1:1,000). The membrane was then cleared in wash
buffer three times for 5 min. Antibody binding was detected using enhanced
chemiluminescence (Millipore, Billerica, MA). Imaging and band quantification were carried
out using a Chemi Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK). Western
blots analyses were determined for 18 participants (n = 6 per group).
Immunoprecipitations: Endogenous AS160 and p70S6K proteins were immuno-
precipitated (IP) overnight at 4°C with 0.8µg of AS160 or p70S6K antibodies in a mix of
protein G-agarose beads (Millipore, Glostrup, DK) and lysate (600µg). The following day
immunocomplexes were washed two times in sucrose lysis buffer and two times in TNE (10
mM Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA and 0.1 mM Na2VO4). The
immunocomplexes were re-suspended in 50 µL of 1 x Laemmli sample buffer and boiled for
5 min (96°C) upon which they were subjected to western blotting as previously described.
Muscle glycogen measurement: Powdered muscle (~20 mg) was hydrolyzed in 250 µl
of 2 N HCl by heating at 95°C for 3 h. The solution was neutralized with 250 µl 2 N NaOH
and the resulting free glycosyl units were assayed spectrophotometrically using a hexokinase-
dependant assay kit (Glucose HK, ABX diagnostics, UK) against glucose standards of known
concentrations (4).
4.3.7 Calculations
Insulin sensitivity: Plasma glucose and insulin concentrations during the 120 min
OGTT were used to determine the whole-body insulin sensitivity index (ISI) according to the
following equation of Matsuda (37):
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149
Matsuda (ISI) = 1000/ √ (FPI·FPG) · (mean OGTT insulin) · (mean OGTT glucose) (1)
Where FPG is the fasting plasma glucose concentration, FPI is the fasting insulin
concentration and 1000 represents a constant that allows numbers between 1 and 10 to be
obtained.
Post-absorptive insulin sensitivity was also estimated by the homeostasis model assessment
(HOMA-IR) index which is calculated by dividing the product of FPG and FPI by 22.5 (38).
Glucose kinetics: From the [2H2] glucose tracer, the total Ra (Eq. 2) and Rd (Eq. 3) of
glucose were calculated with the single-pool non-steady-state equations of Steele (57) as
modified for use with stable isotopes (68). Total Ra represents the splanchnic Ra of glucose
from ingested glucose, the liver and potentially some glycogenolysis and gluconeogenesis
from the kidneys.
Ra total = F - (V· (C2 + C1)/2· (Ep2 – Ep1)/t2 – t1)) / (Ep2 + Epl)/2) (2)
Rd = Ra total – V· (C2 + C1/ t2 – t1) (3)
Where Epl and Ep2 are the [2H2] glucose enrichments in plasma at time-points t1 and t2,
respectively; and C1 and C2 are glucose concentrations at t1 and t2, respectively; and V is
volume of distribution in 160 mL·kg-1
.
The [U-13
C] glucose tracer added to each beverage was used to calculate the Ra of glucose
from the gut. The Ra of [13
C] glucose (Ra gut) into plasma was determined by transposition of
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150
the Steele equation and the known 13
C enrichment of the ingested glucose (52) adapted for use
with stable isotopes (24).
F2 = Ra · [(Ep2 + Ep1)/2 + (C2 + C1)/2· (Ep2 - Ep1)/ (t2 - t1) ·V] (4)
Where F2 is the Ra of [13
C] glucose in the blood; Ra is the previously determined total Ra of
glucose (Eq. 2). Knowing the Ra of [13
C] glucose in the blood, one can determine the
absorption rate of glucose from the gut from the known enrichment of the ingested glucose.
Ra gut = F2/Eing (5)
Where Ra gut is the Ra of gut-derived glucose and Eing is the 13
C enrichment of the ingested
glucose. The rate of endogenous glucose (EGP) was calculated as the difference between Ra
total and Ra gut.
EGP = Ra total - Ra gut (6)
Ra, Rd, Ra gut and EGP were converted to g.min-1
for graphical representation (= µmol·kg-
1·min
-1 x kg x 180.2 / 10
-6).
4.3.9 Statistical Analysis
A between-subject repeated measures design was utilized for the current study.
Exercise variables, blood analytes, plasma enrichment and Western blot data were analyzed
using a two-way ANOVA with repeated measures (treatment x time) to determine differences
between each condition across time. When a significant main effect or interaction was
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151
identified, data were subsequently analyzed using a Bonferroni post hoc test. Plasma glucose
and insulin concentrations over the 120 min OGTT were calculated as area under the curve
(AUC). Within-group changes over time; glucose kinetics and blood analyte AUC data were
checked for statistical significance using one-way repeated-measures ANOVA. All statistical
tests were analyzed using statistical package for social sciences (SPSS) version 18.0 (Illinois,
Chicago, U.S). Significance for all analyses was set at P < 0.05. All values are presented as
means ± standard error of the mean (SEM).
4.4 Results
4.4.1 Dietary intake
Dietary analysis indicated that daily energy intake and macronutrient composition of the
diet was similar for CON, EX and EX+PRO (Table 4.2). Thus, the contents of the
standardized diet provided prior to and during Day 1 of the study were similar for all groups.
CON (n = 8)
EX (n = 8)
EX+PRO (n = 8)
Daily energy intake (kJ) 9,353 ± 469 9,487 ± 691 10,345 ± 452
Carbohydrate (%) 71.1 ± 6.6 66.2 ± 3.9 69 ± 7.1
Protein (%) 15.8 ± 3.0 18.2 ± 3.3 17.9 ± 6.5
Fat (%) 13.1 ± 3.1 15.6 ± 0.4 13.1 ± 4.8
Table 4.2 Participant habitual dietary intake and macronutrient composition.
Groups as per Table 1. Values are presented as means ± SEM.
Chapter 4
152
4.4.2 Exercise variables
Leg-press and leg-extension 1RM values determined during pre-testing, were not
different between groups (Table 4.1). Based on the measured 1RM, the resistance lifted on the
leg-press machine during Day 1 was 150 ± 17 and 156 ± 17 kg and for EX and EX+PRO,
respectively (P > 0.05). Leg-extension resistance was set at 85 ± 11 and 84 ± 9 kg for EX and
EX+PRO, respectively (P > 0.05). All participants were able to complete the leg-press
exercise without reducing the weight. Six participants (four from EX and two from EX+PRO)
were unable to complete the leg-extension exercise at the desired resistance, which was then
lowered by 2.5-5 kg to enable participants to complete ten repetitions. However, the total
exercise volume performed for EX and EX+PRO was not different (Table 4.1).
4.4.3 Plasma glucose
Fasting plasma glucose (5.1 ± 0.4, 5.3 ± 0.7 and 5.7 ± 0.2 mmol·L-1
for CON, EX and
EX+PRO, respectively) were in the normal range. Basal plasma glucose concentrations were
similar on Day 2 (24 h post-exercise) for all groups (5.6 ± 0.3, 4.9 ± 0.3 and 5.1± 0.6
mmol·L-1
for CON, EX and EX+PRO, respectively). During the OGTT, plasma glucose
concentration increased in all groups, peaking between 30-50 min after feeding (P < 0.05;
Figure 4.2A). Plasma glucose peaked at 92 ± 7, 88 ± 9 and 80 ± 8% above basal fasted values
for CON, EX and EX+PRO, respectively. Following the peak, plasma glucose concentration
decreased, such that by 120 min post-OGTT plasma glucose concentration had returned to
basal fasted values. Plasma glucose AUC during OGTT was 17 ± 3% lower for EX and
EX+PRO (P = 0.02 for both) compared with CON (Figure 4.2B).
4.4.4 Plasma insulin
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153
Fasting plasma insulin concentrations (7.2 ± 0.6, 5.9 ± 0.7 and 7.1 ± 0.6 µU·ml-1
for
CON, EX and EX+PRO, respectively) were in the normal range. Basal plasma insulin
concentrations were similar on Day 2 for all groups (6.2 ± 0.7, 6.5 ± 0.4 and 6.9 ± 0.5 µU·ml-
1 for CON, EX and EX+PRO, respectively). Plasma insulin concentrations during OGTT
increased by 8.6-, 9.1- and 11.1-fold above basal fasted values for CON, EX and EX+PRO,
respectively peaking between 30-40 min after feeding (Figure 4.3A). Plasma insulin AUC
during OGTT was significantly greater for EX+PRO compared with EX (P = 0.04) and CON
(P = 0.01). There was no difference in insulin AUC between EX and CON (Figure 4.3B).
Insulin sensitivity: HOMA-(IR) index on Day 1 was 1.55 ± 0.13, 1.69 ± 0.32 and 1.51 ±
0.12 for CON, EX and EX+PRO, respectively. HOMA-(IR) index was not different on Day 2
(1.46 ± 0.17, 1.5 ± 0.12 and 1.64 ± 0.17 for CON, EX and EX+PRO, respectively). Post-
prandial insulin sensitivity, calculated using the Matsuda ISI, was greater for EX and
EX+PRO (6.95 ± 0.5 and 6.82 ± 0.41, respectively) compared with CON (6.21 ± 0.72; P <
0.05). There was no difference in Matsuda ISI between EX and EX+PRO.
4.4.5 Glucose tracer kinetics
Plasma enrichment of infused 6, 6-[2H2] and ingested [U-
13C] glucose are presented in
Figure 4.4A. Ra total, Ra gut, EGP and Rd over time are presented in Figure 4.4 (B, C, D,
respectively). Average plasma glucose tracer kinetics are presented in Table 4.3. In all groups,
plasma glucose Ra total increased over time (P < 0.05), peaking between 70-90 min after
feeding (Figure 4.4B). There was no difference in the plasma glucose Ra total between
groups. Glucose Ra gut demonstrated an increasing contribution to Ra total with time (P <
0.05), whereas EGP demonstrated a reduced contribution to Ra total with time (P < 0.05;
Figure 4.4C). The increase in Ra gut peaked between 70-90 min after feeding, whereas the
Chapter 4
154
decline in EGP reached a nadir between 90-100 min after feeding. Glucose Rd increased over
time in all groups (P < 0.05; Figure 4.4D). Glucose Rd increased by 127 ± 13, 131 ± 15 and
150 ± 18% above basal values for CON, EX and EX+PRO, respectively. Glucose Rd was
significantly lower for CON between 40-70 min after feeding compared with EX and
EX+PRO (P < 0.05). Average glucose Rd and whole-body glucose disposal, (Rd expressed as
% of Ra total) was significantly lower for CON compared with EX and EX+PRO (P < 0.01;
Table 4.3). The time taken for Rd to match the Ra total was greater for CON than EX and
EX+PRO (P < 0.05). There was no difference in average glucose tracer kinetics between EX
and EX+PRO.
4.4.6 Muscle glycogen
Basal muscle glycogen concentration was similar for all groups (Figure 4.5).
Immediately post-exercise, muscle glycogen concentration was 26 ± 8 and 19 ± 6% lower for
EX and EX+PRO, with no difference between groups (P < 0.05). Muscle glycogen
concentration at 24 h post-exercise was similar to basal values for all groups. There was no
significant change in muscle glycogen concentration following OGTT (~26 h post-exercise),
compared with 24 h post-exercise.
4.4.7 Protein phosphorylation
Western blot phospho-images are presented in Figure 4.6. Basal PAS-AS160
phosphorylation (Day 1) was similar for all groups (Figure 4.7A). Compared with basal,
AS160 phosphorylation did not change immediately post-exercise but was increased at 24 h
post-exercise for EX and EX+PRO only (P < 0.05). AS160 phosphorylation at 24 h post-
exercise was significantly greater for EX and EX+PRO compared with CON (P < 0.01). At
26 h post-exercise, following OGTT, AS160 phosphorylation increased by 1.4-fold for CON
Chapter 4
155
and 1.3-fold for EX and EX+PRO compared with 24 h post-exercise (P < 0.05). AS160
phosphorylation at 26 h post-exercise was greater for EX and EX+PRO compared with CON
(P < 0.05). Basal Aktser473
phosphorylation (Day 1) was similar for all groups (Figure 4.7B).
Compared with basal, Akt phosphorylation did not change immediately post- and 24 h post-
exercise in all groups. At 26 h post-exercise, following OGTT, Akt phosphorylation increased
by 2.2-, 2.3 and 1.9-fold for EX and EX+PRO, respectively, compared with 24 h post-
exercise (P < 0.05). Akt phosphorylation at 26 h post-exercise was greater for EX and
EX+PRO compared with CON (P < 0.05). Basal p70S6KThr389
phosphorylation (Day 1) was
similar for all groups (Figure 4.7C). Compared with basal, p70S6K phosphorylation was not
different immediately-, 24 h- or 26 h-post exercise. There was no between-group difference in
p70S6K phosphorylation immediately-, 24 h- or 26 h-post exercise.
Chapter 4
156
CON (n = 8)
EX (n = 8)
EX+PRO (n = 8)
Ra total (g·min-1
) 0.34 ± 0.02 0.36 ± 0.02 0.36 ± 0.03
Ra gut (g·min-1
) 0.24 ± 0.03 0.26 ± 0.03 0.26 ± 0.04
Exogenous contribution
(Ra gut as % of Ra) 65.7 ± 6 68.5 ± 6 65.9 ± 7
EGP (g·min-1
) 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.01
Endogenous contribution
(EGP as % of Ra) 34.2 ± 7 31.4 ± 7 33.9 ± 7
Rd (g·min-1
) 0.31 ± 0.02* 0.35 ± 0.02 0.35 ± 0.02
Glucose disposal (Rd as % of Ra) 89.4 ± 1.7* 94.1 ± 0.5 95.9 ± 0.6
Time for Rd to match Ra (min) 59 ± 8* 42 ± 5 45 ± 6
Ingested glucose appearance (%)
38.4 ± 7.1 41.6 ± 7.6 42.2 ± 7.8
Table 4.3 Plasma glucose kinetics for the 3 groups during OGTT
Data presented for [6, 6- 2H2] glucose rate of appearance (Ra total) and disappearance (Rd) and Rd
expressed as % of Ra. Contribution of exogenous [U-13
C] glucose from the gut (Ra Gut) and
endogenous glucose production (EGP) to Ra total are presented. Groups as per Table 4.1. *
indicates significantly lower than EX and EX+PRO (P < 0.05). Values are presented as means ±
Chapter 4
157
CON EX EX+PRO
Glu
co
se A
UC
(m
mo
l·L
-1 :1
20 m
in)
0
20
40
60
80
100
*
Figure 4.2 Plasma glucose concentration (A) and AUC (B) during OGTT 24 h following
resistance exercise in untrained volunteers. Groups as per Table 4.1. Values are means ±SEM;
n = 8 per group. *: significantly greater glucose concentration/AUC for CON compared with
EX and EX+PRO (P < 0.05).
Time (min)
-60 0 20 40 60 80 100 120
Pla
sm
a g
luco
se
(m
mo
l·L
-1)
0
2
4
6
8
10
12
14 CON
EX
EX+PRO
*
(A)
(B)
Chapter 4
158
Time (min)
-60 0 20 40 60 80 100 120
Pla
sm
a in
su
lin (
µU
·mL
-1)
0
20
40
60
80
100 CON
EX
EX+PRO
*
*
*
†
Figure 4.3 Plasma insulin concentration (A) and AUC (B) during OGTT 24 h following
resistance exercise in untrained volunteers. Groups as per Table 4.1. Values are means ±SEM;
n = 8 per group. *: significantly greater insulin concentration/AUC than CON (P < 0.05). †:
significantly greater insulin concentration than EX. (P < 0.05).
(A)
CON EX EX+PRO
Insulin
AU
C (
µU
·mL
-1:
12
0 m
in)
0
200
300
400
500
600
*
(B)
†
Chapter 4
159
Time (min)
0 20 40 60 80 100 120
MP
E (
%)
0.0
0.5
1.0
1.5
2.0
2.5
3.0 6, 6- [2H
2]
[U-13
C]
a
a a
a
b
b
c
d
(A)
Time (min)
0 20 40 60 80 100 120
Ra T
ota
l (g
·min
-1)
0.0
0.1
0.2
0.3
0.4
0.5
0.6CON
EX
EX+PRO
a a
b
b
b c
a b
c
c c c
b c
b c b c
(B)
Chapter 4
160
Figure 4.4 Tracer enrichments and glucose kinetics over 120 min OGTT. (A) Enrichment of 6,
6-[2H2] and [U-
13C] glucose in plasma. Average for all 3 groups presented, n = 24. MPE (%):
mole percent excess. (B) Total rate of glucose appearance in plasma (Ra). (C) Contribution of
exogenous glucose appearing from the gut (Ra gut) and endogenous glucose production (EGP) to
the Ra Total; solid lines indicate Ra gut, dashed arrows indicate EGP. (D) Rate of glucose
disappearance from plasma (Rd). Means with different subscripts are significantly different from
each other. *: significantly lower Rd for CON compared with EX and EX+PRO. Values are
means ±SEM; n = 8 per group.
Time (min)
0 20 40 60 80 100 120
Ra G
ut
& E
GP
(g
·min
-1)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6 CON
EX
EX+PRO
a
c
c
a
b
b
d
d c , d
d d d
c , d
a
a
a b a , b b b b
b b b
b b
(C)
Time (min)
0 20 40 60 80 100 120
Rd (
g·m
in-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6CON
EX
EX+PRO
*
a a
a
a
b
b
b c
c b c
b c b c
b b
*
*
(D)
Chapter 4. 160
Figure 4.5 Muscle glycogen content. Values obtained at basal, immediately post-exercise
(Post 0), 24 h post-exercise (Post 24) and immediately following OGTT (Post 26). Groups as
per Table 4.1. Values are means ± SEM; n = 6 per group. *: significantly lower compared with
basal (P < 0.05).
Figure 4.6 Representative protein phosphorylation blots. Proteins phosphorylation was studied
in the basal, fasted state (Basal), immediately post-exercise (Post 0), 24 h post-exercise (Post
24) and 26 h post-exercise following the OGTT (Post 26) and normalized to total protein
content.
Basal Post 0 Post 24 Post 26
Mu
scle
gly
co
ge
n (
mm
ol·
kg
-1 w
w)
0
150
200
250
300
350
400
450CON
EX
EX+PRO
*
*
CON
PAS-AS160
T- AS160
AktSer473
T-Akt
p70S6KThr389
T-p70S6K
Basal Post 24 Post 26
EX+PRO
Basal Post 0 Post 24 Post 26
EX
Basal Post 0 Post 24 Post 26
Basal Post 0 Post 24 Post 26
p70S
6K
Th
r38
9 p
ho
pshory
latio
n (
AU
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2 CON
EX
EX+PRO
Basal Post 0 Post 24 Post 26
PA
S -
AS
160
pho
sp
hory
lation
(A
U)
0.0
0.5
1.0
1.5
2.0
2.5CON
EX
EX+PRO
a a
a a
b b
a, b
b
c c
*
*
a, b
Basal Post 0 Post 24 Post 26
AktS
er4
73 p
hosphory
lation (
AU
)
0
1
2
3
4
5
6CON
EX
EX+PRO
a
a
a a
a a
a a
b
b b
*
Figure 4.7 Signalling protein phosphorylation of (A)
Aktser473
, (B) PAS-AS160 and (C) p70S6KThr389
from
muscle samples taken at 4 different time points. Groups as
per Table 4.1. Values are means ± SEM; n = 6 per group.
Means with different letters are significantly different from
each other (P < 0.05). *: significantly lower
phosphorylation for CON compared with EX and EX+PRO
(P < 0.05).
(A) (B)
(C)
Chapter 4 162
4.5 Discussion
The present study expands on the findings of previous investigations (14, 30, 66) by
providing a physiological mechanism to support evidence that a single bout of intense lower-
body resistance exercise (RE) improves insulin sensitivity in healthy, normoglycemic adults.
Using isotopic tracer techniques, we report that prior RE conducted 24 h earlier suppresses
plasma glucose excursions during an oral glucose tolerance test (OGTT) by increasing the rate
of glucose disappearance from the circulation (presumably taken up by skeletal muscle).
Furthermore, we attribute the improvement in insulin-stimulated glucose disposal with prior
RE, in part, to a greater basal and insulin-stimulated phosphorylation of AS160. However, co-
ingesting protein during OGTT did not augment the response of plasma glucose, insulin
sensitivity or glucose kinetics to resistance exercise despite a greater insulin response.
To date, studies investigating the acute effect of RE on insulin sensitivity and glucose
homeostasis have provided conflicting results (5, 14, 15, 30, 33, 66). Whilst questions have
been raised concerning the method used to assess insulin sensitivity (30), the discrepancies
between prior studies may be due, in large part, to; i) the total RE volume, ii) the exercised
muscle group and iii) the physical characteristics of the study cohort. Prior studies utilizing
whole-body RE with a total volume of ~6500 kg (5, 15) showed no effect on plasma glucose
between 12-18 h post-RE, in healthy young (15, 33) and insulin resistant populations (5, 15).
On the other hand, Koopman et al. (30) demonstrated that a lower-limb RE protocol with total
volume ~ 3-fold greater than the aforementioned studies (5, 15), was sufficient to improve
insulin sensitivity in healthy adults. In the present study, we employed an identical RE
protocol Koopman et al. (30) eliciting a similar total RE volume. Accordingly, we too
demonstrated that RE improves insulin sensitivity and lowers post-prandial glucose
Chapter 4 163
excursions at 24 h post-RE. However, we acknowledge that such intense RE may not be
feasible for all insulin resistant/type II diabetic patients. To date, the minimum RE dose
required to counteract symptoms of metabolic disease has not been determined.
In addition to the intensity of exercise, we posit that high-intensity RE confined to the
leg muscles, may promote a greater increase in skeletal muscle glucose uptake than upper-
body RE. Support for this notion comes from a study by Olsen et al. (48), in which, glucose
uptake was found to be relatively well preserved in the arms of Type II diabetics compared
with the legs, indicating that Type II diabetes may be, primarily, a disease of the lower-limbs.
Taken together with the data mentioned above, these findings suggest that a substantial
loading, particularly of the leg muscles, may be an important prerequisite for improved
glucoregulation with RE. Thus, either intensity and/or muscles that are exercised may be
important for improvement of glucose control.
Finally, in combination with RE intensity and muscle groups utilized, the physical
characteristics of the study cohort should be considered when assessing the impact of RE on
glycemic control. For example, Luebbers and colleagues (33) recruited young, resistance-
trained males who habitually completed ≥ 3 RE sessions per week. Participants in this study
completed a whole-body RE protocol at high (85% of 10RM) and low (45% of 10RM)
intensities, matched for total volume (~12,600 kg). Compared with a non-exercise control
trial, the authors reported no effect of low-, and more surprisingly, high-intensity RE on
glucose uptake and insulin sensitivity during a hyperinsulinemic-euglycemic clamp. In
contrast, others (14) have shown that normoglycemia is better maintained in Type II diabetics
who completed a whole-body RE protocol (80% of 3RM) similar to that employed by
Luebbers (33). Taken together, these data suggest that the physical characteristics of the study
Chapter 4 164
cohort must be taken into account when prescribing an exercise volume intended to induce
sufficient ‘stress’ across the exercised muscle to lower blood glucose.
The 17% reduction in plasma glucose response we observed with prior RE is in line
with previous studies (14). However, modifications in blood analytes do not necessarily
reflect adaptations in skeletal muscle glucose uptake. Therefore, we applied dual isotopic
tracer methodology to determine the rate of glucose disappearance from the circulation
(indicative of skeletal muscle glucose uptake (9)) and the contribution of exogenous glucose
appearance and endogenous glucose production to total glucose appearance. Total glucose
appearance and the contribution from endogenous and exogenous sources was not different
between groups, indicating that gastric emptying, intestinal uptake and hepatic glucose output
were not altered by prior RE or protein co-ingestion during OGTT. However, plasma glucose
disappearance/disposal from the circulation did increase with prior RE. Further, we calculated
that the time taken for glucose disappearance to match glucose appearance was reduced by
~37%. Thus, RE may be potent intervention to reverse the compromised insulin-stimulated
glucose disposal that is prevalent in Type II diabetes (67). Despite the greater increase in
glucose disposal in all groups during OGTT we report no increase, or between-group
difference, in post-OGTT muscle glycogen content. This may not be surprising given that the
amount of ingested glucose disposed from the circulation over 120 min of OGTT was not
vastly different between exercised (~55%) and non-exercised groups (~50%).
Enhancement of glucose disposal with prior exercise appears to be mediated through the
insulin signalling pathway. A number of studies have found that prior exercise has no effect
on proximal insulin signalling steps, such as Akt phosphorylation (1, 20). Similarly, we report
no effect of RE on basal Akt phosphorylation at 24 h post-exercise. In contrast, the Rab-
Chapter 4 165
GTPase-activating protein AS160 is the most distal signalling protein implicated in insulin-
stimulated glucose uptake in skeletal muscle. Studies in humans (12, 21) demonstrate that
basal and insulin-stimulated PAS-AS160 phosphorylation is increased several hours post-RE.
Until now, the sustained effect of prior exercise on PAS-AS160 phosphorylation has been
demonstrated in animals only (1, 16). Here we demonstrate for the first time, in humans, that
immunoprecipitated PAS-AS160 phosphorylation is greater at 24 h post-RE compared with
basal and non-exercise values. Moreover, although the insulin-stimulated rise PAS-AS160
phosphorylation was similar for all groups, the final level of PAS-AS160 phosphorylation
was greater for EX and EX+PRO compared with CON following the OGTT.
Important to note is that not all studies report similar adaptations in PAS-AS160
phosphorylation with prior exercise. Treebak and colleagues (60) have shown that basal and
insulin-stimulated site-specific, but not PAS, phosphorylation of AS160 increased 4 h after a
60 min unilateral leg kicking protocol. Given that we and others observed a marked increase
in basal PAS-AS160 phosphorylation following high-intensity RE loading, we speculate that
the PAS-AS160 antibody may only be effective in detecting robust modifications in AS160
phosphorylation, whereas site-specific AS160 phosphorylation may be required to detect
more subtle phospho-changes that occur with moderate-intensity exercise. Thus, regardless of
the antibody used to detect AS160 phosphorylation our data expand human work (12, 21) and
support animal studies (1, 16) showing the persistent increase in insulin-stimulated skeletal
muscle glucose transport following acute RE is attributable, in part, to the sustained increase
in basal AS160 phosphorylation. These data do not exclude the possibility that prior exercise
also amplifies insulin-signalling steps other than AS160.
Chapter 4 166
To our knowledge, this study is the first to attempt to determine if protein co-ingestion
augments the response of RE on glycemic control at 24 h post-exercise. Under resting
conditions, it has been demonstrated that the marked increase plasma insulin concentration
that prevails when protein and/or amino acids are ingested with carbohydrate can effectively
increase glucose disposal (36) and reduce plasma glucose excursions (62, 65). In our hands,
the elevated insulin response with protein co-ingestion did not augment the RE-induced rise in
glucose disposal or lower plasma glucose excursions. These data are supported by the
response of the intracellular signalling indicating that although the final level of PAS-AS160
phosphorylation was greater with prior exercise, there was no difference with protein
ingestion. We posit that the lack of a glycemic-lowering effect of additional protein could be
attributed to the insulin response to our feeding protocol. Our data reveal that the transient rise
in plasma insulin with additional protein peaked at 30 min post-feeding (21 and 13% greater
than CON and EX, respectively) but was no longer evident by 60 min. In contrast, studies that
report a glucose lowering effect with protein co-ingestion have favoured frequent feeding of
small boluses to promote a sustained rise in plasma insulin, much greater than the present
study (36, 62, 63, 65). For example, participants in the study of Manders et al. (36) ingested
beverages every 15 min over 165 min supplying ~60g·h-1
carbohydrate alone or with ~30g·h-1
of additional protein. The greater plasma insulin response that prevailed with additional
protein was sustained for the best part of 3 h. In contrast, the greater insulin response we
observed with additional protein was relatively transient (between 30-50 min post-feeding).
Thus, this relatively brief period of hyperinsulinemia may have been insufficient to further
lower plasma glucose excursions. However, this approach has not been tried following
resistance exercise.
Chapter 4 167
A limitation of our protocol and that of other studies (36, 62, 63, 65) is that feeding
high-glucose loads and/or repeated boluses does not represent a physiological feeding pattern
of Type II diabetic populations. Conversely, the group of Nuttall and Gannon (17, 18, 26, 27,
45, 47) systematically investigated the insulinotropic properties of ≈ 5g of essential (arginine,
phenylalanine, lysine and leucine) and non-essential amino acids (glycine, proline) co-
ingested with 25g of glucose, closely mimicking the composition of a high-protein meal. By
and large, these studies demonstrate that free amino acid co-ingestion initiates only a modest
rise in insulin, but markedly attenuates the plasma glucose response. Combining these data
with our observation that protein co-ingestion with a high glucose load did not lower blood
glucose, one could suggest that co-ingesting specific free amino acids with glucose in an
amount likely to be ingested during a typical meal, may lower plasma glucose excursions via
subtle changes in plasma insulin or direct mechanisms acting independently of the insulin.
The lack of a glucose-lowering effect with protein co-ingestion could also be explained
by the relatively modest leucine content (~2.65g) in the 25g of intact whey protein we
provided. A number of studies have demonstrated that certain free amino acids can act as
potent insulin secretagogues (53, 54). Indeed, a series of studies (31, 63-65) revealed that
adding ~7.5g of free leucine to a protein hydrolysate plus carbohydrate beverage augmented
the insulinotropic response. Subsequently, this mixture was shown to suppress post-prandial
glucose excursions in Type II diabetics (35, 62). In vitro studies indicate this response may be
due to the potency of leucine in secreting insulin release from pancreas β cells (55, 69).
Furthermore, there is mounting evidence in animal and cell culture models, that leucine
directly promotes glucose transport into skeletal muscle by increasing GLUT4 translocation
via the mammalian target of rapamycin (mTOR) signalling pathway (2, 43, 51). Thus, given
that we saw no change in p70S6K phosphorylation (downstream of mTOR) for EX+PRO, it is
Chapter 4 168
possible that ingesting additional free amino acids, leucine in particular, are essential to
facilitate glucose uptake in skeletal muscle and disposal from the circulation. Further work is
required to determine the effectiveness of ingesting specific free amino acids, particularly
leucine, in combination with RE for improving glycemic control.
Finally, it is worth mentioning that the studies discussed above, reporting a glucose-
lowering effect of protein co-ingestion, were conducted under resting conditions. In contrast,
we report no additive effect of protein co-ingestion, perhaps due to the fact that the
glucoregulatory effects of feeding were assessed following high-intensity RE. Thus, we
speculate that the robust increase in basal PAS-AS160 phosphorylation with prior exercise
may have masked any additive effect of additional protein/insulin. Indeed our signalling data
indicate that the insulin-stimulated rise in PAS-AS160 phosphorylation during OGTT was
similar for all groups. Based on these data, it is unclear whether a synergy exists between RE
and protein ingestion for promoting hypoglycemia.
In conclusion, we have shown that high-intensity resistance exercise improves insulin
sensitivity and increases the rate of postprandial glucose disposal, which subsequently lowers
post-prandial glucose excursions in healthy, normoglycemic adults. However, despite strong
evidence that co-ingesting protein and/or amino acids with carbohydrate lowers glucose
excursions at rest, we show no effect of protein co-ingestion on glycemic control, despite a
marked rise in insulin secretion. Thus, we hypothesize that the co-ingestion of additional free
amino acids and/or sustained hyperinsulinemia may be essential in order to increase skeletal
muscle glucose uptake.
Acknowledgements
Chapter 4 169
The authors would like to thank Darren Briscoe, Amber Cottam, James Gilbert and
Lorna Webb for their assistance during data collection and Jinglei Yu for technical assistance.
We also thank Professor Graeme Hardie at the University of Dundee for supplying antibodies.
We extend our appreciation to the participants for their time and effort. This work was
supported by a grant from The Insulin Dependent Diabetes Trust to K. D. T.
Author contributions
L.B., A. P., C.S.S., A.E.J., K.B and K.D.T contributed to the conception and design of
the experiment. L.B., A.P., C.S.S., K.B. and K.D.T. contributed to the collection of the data.
L.B., A.P., C.S.S., K.B and K.D.T. contributed to the analysis and interpretation of the data.
L.B., A.P., C.S.S., K.B and K.D.T. contributed to drafting or revising the content of the
manuscript.
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Chapter 5 175
CHAPTER 5
GENERAL DISCUSSION
Several studies have demonstrated that ingesting protein with carbohydrate during
exercise can extend endurance capacity (20, 37, 38) and improve late-exercise performance
(39). In the post-exercise period, protein ingestion may also impact the metabolic adaptation
in skeletal muscle and potentially improve the recovery of muscle function. However,
concerns regarding the way in which exercise performance and recovery are assessed,
coupled with the lack of a clear physiological mechanism to explain the reported
improvements, hampers our understanding of the role of protein nutrition for endurance
athletes. Beyond athletic performance, there is great interest in modulating skeletal muscle
metabolism to counter insulin resistance and Type II diabetes. Indeed resistance exercise has
been demonstrated to improve whole-body insulin sensitivity (8, 23) and glycemic control
(7), yet the mechanisms underpinning this adaptive response remain elusive. Furthermore, the
notion that protein nutrition can help to maintain normoglycemia is yet to be investigated in
combination with resistance exercise. Thus, the purpose of this thesis was three-fold:
i) To determine the effectiveness of co-ingesting protein with carbohydrate during
exercise on endurance performance and the recovery of muscle function.
ii) To determine the impact of post-endurance exercise protein ingestion on the
synthesis of specific skeletal muscle proteins.
iii) To understand the mechanisms underpinning improved glycemic control following
resistance exercise and investigate whether ingesting additional protein augments
this response.
Chapter 5 176
The present chapter summarizes the key findings from the studies described in
Chapters 2-4 of this thesis. First, based on the findings described in Chapters 2 and 3, this
section will provide recommendations for future research investigating the effectiveness of
protein ingestion for endurance performance and changes in protein metabolism that, over
time, may quicken the recovery of muscle function and alter the phenotypic adaptation to
training. Second, based on the findings described in Chapter 4, this section will provide
recommendations for future research studies expanding on our investigation of the
effectiveness of resistance exercise, with or without protein ingestion, on glycemic control.
5.1 Protein nutrition for endurance performance
Over the last decade a number of studies have emerged to suggest that the co-ingestion
of protein with carbohydrate during prolonged endurance exercise can extend endurance
capacity (20, 37, 38). Furthermore, others have reported that protein co-ingestion specifically
improves late-exercise time-trial performance (39). However, methodological differences in
the performance test employed in previous studies makes it difficult to determine whether
adding protein to carbohydrate improves endurance performance per se or merely time-to-
exhaustion. In support of this notion, several studies report no ergogenic benefit of
carbohydrate plus protein co-ingestion on time-trial performance in a well-controlled testing
environment (33, 44). Furthermore, when sufficient carbohydrate is ingested the role of
protein for improving endurance performance may be negligible (44). In an attempt to
improve our understanding of the role of protein for endurance athletes, the logical extension
of these findings was to examine the effectiveness of protein co-ingestion using a
Chapter 5 177
performance model that replicates endurance competition and a carbohydrate dose that meets
the recommended upper-limit to attain peak exogenous carbohydrate oxidation rates.
Thus, in our study (Chapter 2) we provided a recommended dose of carbohydrate (65
g·hˉ1) during cycle exercise to determine whether the addition of protein (19 g·hˉ
1) improved
cycle time-trial performance in a controlled exercise environment, when preceded by a
standardized steady-state ride. Our data demonstrate no difference in average power output
during the time-trial or overall time-to-completion between carbohydrate plus protein and
carbohydrate alone. Thus, these data oppose the notion that carbohydrate-protein co-ingestion
during exercise enhances late-exercise performance when sufficient carbohydrate is ingested.
5.1.1 Future studies
In the study described in Chapter 2, we examined the effectiveness of adding protein
to a carbohydrate-containing beverage on late-exercise time-trial performance and the
recovery of muscle function. Our decision not to match treatment beverages for total energy
was based on the observation that others report an extended endurance capacity when protein
was co-ingested with a carbohydrate dose deemed sub-optimal to attain peak rates of
exogenous carbohydrate oxidation (20, 37, 38). Thus, our findings support those of van-Essen
et al. (44), indicating that there is no benefit of co-ingesting additional protein in a sports
beverage on time-trial performance provided sufficient carbohydrate is consumed. However,
the metabolic and physiological response to carbohydrate-protein co-ingestion may be
dependent on the mode, duration and intensity of the exercise bout. In this regard there is
evidence that protein oxidation is increased when protein is ingested with a carbohydrate
Chapter 5 178
intervention during ultra-endurance exercise (24). However, the effectiveness of protein co-
ingestion for performance, in situations where carbohydrate availability is limited and muscle
glycogen stores are depleted has yet to be investigated, but would prove interesting given
evidence from almost 20 years ago that runners ingesting branched-chain amino acid alone
took less time to complete a marathon (2).
5.2 Muscle adaptation to acute endurance exercise and protein nutrition
Endurance exercise stimulates an increase in mixed muscle protein synthesis. This
response has been shown to result primarily from a rise in the synthesis of mitochondrial
proteins, with little or no change reported in the synthesis of myofibrillar proteins (51). Given
evidence that ingesting protein and/or amino acids potentiates resistance exercise-stimulated
muscle protein synthesis, investigators have suggested that protein nutrition may enhance the
synthesis of mitochondrial proteins following endurance exercise (19). Our study (Chapter
3) examined the response of mitochondrial and myofibrillar protein fractions to protein
ingestion following endurance exercise. We report that myofibrillar protein synthesis was
greater when protein was ingested with a carbohydrate beverage (~0.087%·h-1
) than
carbohydrate ingested alone (~0.057%·h-1
). Conversely, protein ingestion did not stimulate
an increase in mitochondrial protein synthesis. Thus, we are able to attribute the increase in
mixed muscle protein synthesis demonstrated by Howarth et al. (19) to a rise in the synthesis
of myofibrillar, but not mitochondrial proteins.
Although the primary aim of our study was to determine the synthesis of specific
protein fractions, in collaboration with Drs Keith Baar and Andy Philp of the University of
Chapter 5 179
California, Davis, we were able to measure the phosphorylation of signalling proteins
implicated in the translation initiation and elongation phase of muscle protein synthesis
immediately and 4 hours post-exercise for 8 participants. In spite of the fact that some of the
anabolic signalling response may have occurred between 0 and 4 hours post-exercise, we
demonstrate that the phosphorylation of intracellular proteins was increased (mTOR,
p70S6K) or was suppressed (eEF2) following protein ingestion, thereby providing a
mechanism to support our muscle protein synthesis data. Collectively, these data suggest that
when protein is ingested following intense cycling exercise contractile proteins are
preferentially synthesized in the initial 4 hour post-exercise period.
5.2.1 Future studies
An acute increase in myofibrillar protein synthesis, with protein ingestion and exercise,
is predictive of long-term muscle hypertrophy (17, 52). Thus, one interpretation of the data
presented in Chapter 3 is that protein ingestion following prolonged high-intensity cycling
over an extended period may increase lean mass. However, the rise in myofibrillar protein
synthesis we observed post-exercise, may serve to counteract a fasted-state rise in protein
breakdown thought to occur during and following intense endurance exercise (3, 12, 45).
Thus, the rise in myofibrillar protein synthesis may represent an increase in protein turnover;
important in the maintenance of a higher quality of contractile protein. However, we did not
measure protein breakdown and thus, this notion is speculative at this point. Thus, it would be
prudent for future studies to ascertain changes in muscle protein breakdown (and proteins
implicated in proteolysis) during and following endurance exercise with protein feeding.
Finally, given that resistance-exercise performed in the fed-state elevates muscle protein
synthesis (54) over that performed when fasted, it is likely that the adaptive response to
Chapter 5 180
protein ingestion after endurance exercise would be dramatically altered by prior feeding.
This approach may also represent, more closely, common practice amongst athletes and
warrants further investigation. Based on data from previous studies (3, 12, 16, 45) and our
observation that myofibrillar proteins are preferentially synthesized when protein is ingested
after endurance exercise, we present herein the proposed temporal response of myofibrillar
net protein balance over the course of the carbohydrate plus protein trial (Figure 5.1).
In our study, muscle protein synthesis was assessed over 4 hours post-exercise. The
response of mitochondria to endurance exercise is a transcriptionally mediated process and
occurs later than the time-course of our investigation (11, 18, 40). Thus, a logical extension to
our findings would be to investigate the response of myofibrillar and mitochondrial protein
synthesis to endurance exercise and protein ingestion beyond 4 hours post-exercise. Such an
approach would improve our understanding of the muscle adaptations that occur when
protein is ingested after endurance exercise. Furthermore, to resolve the precise role of
protein nutrition for endurance athletes we suggest that longitudinal studies must be
conducted, in which, protein is ingested post-exercise over the course of a training regimen to
determine the chronic phenotypic and physiological adaptations.
Other possibilities to explain the lack of change in mitochondrial muscle protein
synthesis is that the training status of the subjects and the exercise protocol utilized in the
present study. It has previously been demonstrated that the response of mitochondrial protein
synthesis to an acute bout of cycling tends to be lower in individuals after 10-weeks of
endurance training (51). Thus, our data may suggest that the synthetic response of
mitochondrial proteins in well-trained individuals reaches a ‘ceiling’; the point beyond
Chapter 5 181
% h
-1
-0.12
-0.09
-0.06
-0.03
0.00
0.03
0.06
0.09
0.12
Exercise
C+P
-NBAL
+NBAL
Basal Post 0 Post 60 Post 240
which, further contractile and/or nutrient stimuli fail to augment the synthetic response. Thus,
future studies should seek to characterize the response of different protein fractions to
endurance exercise in cohorts with divergent phenotypes (i.e. untrained healthy, untrained
obese, resistance trained).
5.3 Protein nutrition for recovery from endurance exercise
Although we have demonstrated that there is no effect of carbohydrate-protein co-
ingestion on endurance exercise performance, the findings of Chapter 3 indicate that protein
ingestion after endurance exercise has a marked influence on the synthesis of contractile
Figure 5.1 Proposed time-course of net myofibrillar protein balance following endurance
exercise and protein feeding. Temporal swing of negative (-NBAL) and positive (+NBAL)
net myofibrillar protein balance throughout the protocol described in Chapter 3.
Chapter 5 182
proteins and this may play an important role in the recovery process. The increase in muscle
protein synthesis we and others (19, 25) demonstrate when protein is ingested after endurance
exercise has been suggested as a potential mechanism to explain how protein ingestion
attenuates indirect markers of ultra-structural damage such as, for example, plasma creatine
kinase concentration (26, 29, 36-38, 41, 43). Ultimately, these acute adaptations are thought
to improve the subsequent rate of recovery; defined as the recovery of endurance capacity
(37, 53) or muscle function (41, 43).
During the study outlined in Chapter 2, participants returned to the laboratory 24 hours
post-exercise to determine whether the additional protein ingested during the initial exercise
bout ameliorated muscle soreness, plasma creatine kinase concentration and improved the
recovery of maximal isometric strength. As expected, we observed a rise in plasma creatine
kinase and perceived muscle soreness and a resultant decline in isometric strength at 24 hours
post-exercise. However, the additional protein did not attenuate indices of muscle damage or
improve the recovery of isometric strength. Thus, despite evidence in Chapter 3 indicating
that ingesting additional protein in the post-exercise period increases the synthesis of
structural proteins, we demonstrate no effect of protein ingestion during exercise on any of
the indices of recovery we measured.
5.3.1 Future studies
The effectiveness of protein nutrition as a means to improve the rate of recovery is
likely hampered by time lapse between exercise completion and the assessment of recovery.
Reported changes in muscle damage with protein ingestion are typically demonstrated several
Chapter 5 183
hours after exercise (37, 53). Despite the robust acute increase in myofibrillar protein
synthesis we observed, the complete turnover of myofibrillar proteins (i.e. incorporated into
the myofiber) is relatively slow; on the order of days-to-weeks (34, 35). Thus, it is difficult to
comprehend how this process may influence changes noted in just a few hours. These data
may explain why we and others (13) were unable to detect any influence of protein ingestion
on recovery between 24 hours post-exercise. Therefore, based on our combined findings in
Chapters 2 and 3, it would prove useful for future studies to examine recovery in the
immediate hours through to several-days post-exercise.
The lack of conformity between the data presented may also be explained by the
different feeding protocols employed. The absence of any improvement in recovery (Chapter
2) was demonstrated when protein was ingested during prior exercise. Conversely, the
increase in myofibrillar protein synthesis we report (Chapter 3) occurred when protein was
ingested following exercise. Given that no study has investigated the influence of
manipulating the timing and/or frequency of carbohydrate-protein co-ingestion on subsequent
rates of recovery, this avenue of investigation is important in order to shed further light on the
effectiveness of protein nutrition for endurance athletes.
5.4 Metabolic adaptation to acute resistance exercise and protein nutrition
Acute resistance exercise can improve glycemic control in healthy (23) and Type II
diabetic individuals (7) via insulin-independent mechanisms (6, 42). Further, co-ingesting
protein and/or amino acids with carbohydrate loads potentiates the release of insulin,
reducing the prevailing plasma glucose response (28, 48). Thus, resistance exercise and
Chapter 5 184
protein nutrition represent safe alternatives to pharmacological interventions to alleviate or
reverse the manifestations of Type II and its co-morbidities.
Our study (Chapter 4) examined the effectiveness of an intense lower-limb resistance
exercise bout on glycemic control during a glucose challenge at 24 hours post-exercise. One
group of participants co-ingested additional protein during the glucose challenge to determine
whether protein feeding would augment the exercise-induced changes in glucoregulation.
Using a dual stable isotopic tracer technique during the glucose challenge, we demonstrated
that prior exercise increased the rate of glucose disposal from the circulation by ~12%, which
ultimately, lowered post-prandial plasma glucose excursions by ~17% compared with non-
exercised controls. At the skeletal muscle level, we demonstrate that prior exercise increased
basal PAS-AS160 phosphorylation. These data suggest that greater basal AS160
phosphorylation with prior exercise enhances insulin-stimulated glucose uptake, as evidenced
by the greater rate of glucose disposal and the final level of PAS-AS160 phosphorylation
following OGTT. Interestingly, protein co-ingestion during the glucose challenge did not
augment the exercise-induced improvement in glycemic control or modify signalling protein
phosphorylation, despite an elevated insulin response in this sub-group.
5.4.1 Future studies
The high-intensity exercise model utilized in Chapter 4 was adopted based on data
suggesting that a substantial total exercise volume is required in order to promote a beneficial
effect on glucose homeostasis (4, 8, 23, 27), particularly in non-diabetic individuals. Whilst
the total exercise volume in our study and that of Koopman et al. (23) was ~3-fold greater
than studies demonstrating no glucose-lowering effect of resistance exercise (4, 8, 27), the
Chapter 5 185
fact that exercise was restricted to the lower-limbs may also have contributed to the reduction
in blood glucose. Indeed, Olsen et al. (32) have shown that Type II diabetes may, primarily,
be a disease of the lower limbs (32). Thus, investigating the chronic effects of lower-limb
versus whole-body resistance training regimens on glucose homeostasis and insulin
sensitivity in Type II diabetics would be an interesting route for future studies. Furthermore,
although a growing body of experimental evidence suggests that long-term resistance training
improves metabolic homeostasis in a manner distinct from endurance training; our
understanding of how this precisely occurs is rudimentary. Thus, additional research is
clearly needed to better understand how different modes of physical training (i.e., resistance,
endurance or an interval exercise) change the metabolic and qualitative characteristics of
skeletal muscle and improve glycemic control. Finally, given that the typical physiological
characteristics of Type II diabetics are dramatically different from young, healthy,
normoglycemic males we recruited, the high-intensity resistance exercise protocol in our
study may not be feasible for Type II diabetic individuals to complete. Thus, future studies
should seek to determine the minimum ‘dose’ of resistance exercise required to counteract
symptoms of metabolic disease.
In Chapter 4 we demonstrated that basal and PAS-AS160 phosphorylation was
increased with prior exercise. Thus, the elevated glucose disposal with prior exercise can be
attributed, in large part, to an increase skeletal muscle glucose uptake mediated by AS160.
However, this does not exclude the possibility that prior exercise also amplifies insulin-
signalling steps other than AS160. In addition, we and others (1, 15) report no effect of prior
exercise on the basal phosphorylation of AktSer473
. Interestingly, we did observe an increase
in insulin-stimulated Akt phosphorylation with prior exercise. Thus, future studies should
seek to characterize, in greater detail, basal and insulin-stimulated modifications in insulin-
Chapter 5 186
signalling intermediates following acute resistance exercise. Finally, it should be noted that
this study presents a limitation due to the number of the participants analyzed for protein
phosphorylation (6 per group). Thus, due to the small sample size and insufficient statistical
power, it is possible the analyses may have contained a type 2 error. As a consequence, the
lack of statistical significance in proximal insulin signalling intermediates should be taken
with caution.
Under resting conditions, the insulinotropic response of co-ingesting protein with
carbohydrate is thought to facilitate the reduction in plasma glucose excursions (46, 49). In
Chapter 4, we show that, despite an elevated insulin response, protein co-ingestion during a
glucose challenge did not augment the beneficial effect of resistance exercise on plasma
glucose disposal or glucose excursions. We posit that this null finding may be due to; i) the
relatively brief period of hyperinsulinemia with protein co-ingestion, ii) the absence of
specific amino acids or iii) the fact that the robust effect of prior exercise on insulin signalling
and glucose disposal may have masked an additive effect of protein/insulin. Indeed, the
hypoglycaemic effect of protein co-ingestion has been demonstrated with different feeding
protocols to that used in our study. In summary, these data suggest that a period of sustained
hyperinsulinemia (28, 46, 47, 49) or specific free amino acids (9, 10, 21, 22, 30, 31) may be
important in order for protein ingestion to lower blood glucose concentration. Given the
potency of protein ingestion for maintaining glucose homeostasis, further studies should
investigate the effect of different protein feeding strategies (for example, frequent feeding
and branched-chain amino acid ingestion) following resistance exercise to ascertain whether
protein and/or amino acids can augment the metabolic benefits we have found.
Chapter 5 187
Finally, it would be remiss not to mention that ingesting protein and/or amino acids
confers other benefits to metabolic health benefits besides glucoregulation. In addition to
hyperinsulinemia, co-ingesting protein with carbohydrate promotes hyperaminoacidemia and
has been found to stimulate protein synthesis and inhibit protein breakdown (14, 50). Thus,
ingesting amino acids following resistance exercise may blunt the markedly elevated muscle
protein breakdown rates commonly found in uncontrolled Type II diabetes (5). Future studies
should seek to clarify this hypothesis.
5.5 Conclusions
In this thesis we have demonstrated that protein nutrition during exercise does not
improve cycle time-trial performance, attenuate purported markers of muscle damage or
enhance the recovery of muscle strength. Alternatively, protein ingestion following
endurance exercise can affect the acute response of intracellular signalling proteins
implicated in mRNA translation, thereby increasing the synthesis of contractile muscle
proteins. Thus, over time this acute adaptive response may; i) promote muscle hypertrophy or
ii) confer benefits for muscle recovery and regeneration. However, it is possible that an
increase in mitochondrial protein synthesis may occur at a later time-point in the recovery
process.
Beyond athletic performance and adaptation, data presented in this thesis demonstrate
that a single bout of high-intensity resistance exercise increases insulin-stimulated glucose
uptake, lowering plasma glucose concentration during feeding 24 hours post-exercise. The
mechanism accounting for this action appears to involve a greater phosphorylation of distal
Chapter 5 188
insulin-signalling intermediates that subsequently promote an increase in insulin-stimulated
glucose transport in skeletal muscle. However, in this study the ingestion of additional
protein did not impact the signalling response in skeletal muscle or improve glycemic control.
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Chapter 5 189
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ingestion markedly attenuates the glucose response to ingested glucose without a change in
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Chapter 5 190
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Postexercise protein intake enhances whole-body and leg protein accretion in humans. Med
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26. Luden ND, Saunders MJ, and Todd MK. Postexercise carbohydrate-protein-
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27. Luebbers HT, Messmer P, Obwegeser JA, Zwahlen RA, Kikinis R, Graetz KW,
and Matthews F. Comparison of different registration methods for surgical navigation in
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28. Manders RJ, Wagenmakers AJ, Koopman R, Zorenc AH, Menheere PP,
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acid mixture with carbohydrate improves plasma glucose disposal in patients with type 2
diabetes. Am J Clin Nutr 82: 76-83, 2005.
29. Millard-Stafford M, Warren GL, Thomas LM, Doyle JA, Snow T, and
Hitchcock K. Recovery from run training: efficacy of a carbohydrate-protein beverage? Int J
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proline with and without glucose. Metabolism 53: 241-246, 2004.
31. Nuttall FQ, Schweim KJ, and Gannon MC. Effect of orally administered
phenylalanine with and without glucose on insulin, glucagon and glucose concentrations.
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in arm than leg muscle in type 2 diabetes. J Physiol 565: 555-562, 2005.
33. Osterberg KL, Zachwieja JJ, and Smith JW. Carbohydrate and carbohydrate +
protein for cycling time-trial performance. J Sports Sci 26: 227-233, 2008.
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of the human muscle mass. Annu Rev Physiol 66: 799-828, 2004.
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protein turnover: the concept of anabolic resistance and its relevance to the transplant
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Chapter 5 191
38. Saunders MJ, Luden ND, and Herrick JE. Consumption of an oral carbohydrate-
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97: 557-567, 1999.
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Chapter 5 192
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Chapter 6 193
CHAPTER 6
APPENDICES - METHOD DEVELOPMENT
6.1 Method of mitochondrial and myofibrillar protein isolation
Studies measuring abundant skeletal muscle protein fractions, for example, myofibrillar
and sarcoplasmic proteins have been relatively forthcoming since methods to separate out
these fractions were developed in the late 1990s. Yet, studies reporting the effects of
interventions on mitochondrial protein synthesis have been elusive. This apparent
underreporting of mitochondrial protein synthesis is likely due to technical difficulties rather
than a lack of aspiration by researchers in this area. Indeed, the amount of mitochondrial
protein from rat skeletal muscle yields ~1 and 2-4 mg/g muscle wet weight of sub-
sarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, respectively (5). Most human
muscle biopsies taken from the vastus lateralis muscle weigh ~100-200 mg wet. Therefore,
the expected yield of mitochondrial protein from muscle biopsy tissues would be < 1 mg.
This amount of protein is challenging to measure using current GCMS and GC-C-IRMS
methods. Therefore, it is not surprising that limited research is available on the regulation of
mitochondrial protein synthesis with exercise or nutrient provision.
There are two sub-populations of mitochondria in the muscle; IMF mitochondria are
located between myofibrils and SS mitochondria are located beneath the sarcolemma. SS
mitochondria represent 20% of mitochondria within skeletal muscle and IMF mitochondria
account for the remaining 80% (2). In humans, both populations of mitochondria have been
Chapter 6 194
shown to adapt to endurance exercise training by increasing in number (3, 4). Until recently,
studies in humans had only characterized the rates of protein synthesis of SS mitochondria at
rest (1, 6). In one of these studies, Rooyackers et al. (6) stated that they were unable to obtain
the IMF mitochondria because the proteolytic enzymes required to do so interfered with the
measurements of the rate of synthesis of myofibrillar proteins. More recently, Wilkinson et
al. (7) characterized the synthetic response of mitochondrial proteins to different modes of
exercise by combining both IMF and SS mitochondrial fractions obtained during sample
processing.
In Chapter 3 of this thesis, we adapted the method of protein isolation from Wilkinson
et al. (7). This method utilizes techniques of muscle tissue homogenization and centrifugation
to separate cellular components of different densities. The principle underlying this technique
is that the heaviest (or most dense) cell components pellet in less time and with less force
than is required to pellet lighter organelles such as mitochondria. Therefore, once the tissue
homogenate is obtained, a first slow speed spin (~1000g for 10 min) is used to pellet the
myofibrillar protein. The supernatant is then removed and transferred to another tube and
centrifuged at a higher speed (~10,000g for 10 min) to pellet the lighter mitochondria. Prior
to study analyses, we confirmed (in human tissue from pilot studies) that this method was
appropriate to obtain both mitochondria and myofibrillar proteins from a mixed muscle
homogenate.
6.1.1 Method preparation and supplies
• Tissue needed: Possible with 40-50mg but better with 70-80mg.
• 4mL black screw top glass vials.
• Coated razorblade to divide muscle in N2.
Chapter 6 195
• Teflon (polypropylene) pestles (Schutt Labortechnik GmbH: Product # 3.200.512).
• Steel dish to store samples in N2.
• 1.5mL Eppendorf.
• Pipette tips (blue, yellow, pastette pipettes).
• Glass culture tubes (Fisher scientific 10x75mm: Product # 14-961-25).
• Glass Dounce homogenizer with glass pestle x 2.
6.1.2 Sample preparation prior to isolation
• When weighing the sample always keep on N2 or dry ice.
• Use fresh 1.5mL eppendorf to zero the balance. Once sample (in cryotube) is out of
duer and into dish of N2 (or dry ice) open cap and tip muscle out into the N2.
• Using razorblade/scalpel, chip the frozen muscle to weigh out very specific amounts
then add to 1.5mL eppendorf.
• Discard old cryotube.
6.1.3 Reagent preparation
• 0.3 M Sodium hydroxide (NaOH)
• 95% Ethanol (EtOH)
• 6 M Hydrochloric acid (HCl)
• 1 M Perchloric acid (PCA)
• β-2-glycerophosphate (Sigma: Product # G9891-25G)
• MgCL (Sigma 99.0 – 102%: Product #M9272-100G)
• NaFl (Sigma: Product # 51504-100G)
• Tris base / Tris HCl (Sigma: Product #T6066-1KG / Sigma: Product # T5941-500G)
• EDTA (Siga: Product # E9884-100G)
• KCl (Sigma 99%: Product # P4505-1KG)
Buffer is made up into 20mL of 50mM Tris buffer. Tris buffer contains 6.75g/L of Tris HCl
and 1.18g/L of Tris base for 0.05 M solution. The following reagents are weighed and placed
into 50mL falcon tube with 20mL of Tris buffer added on top.
.
Chapter 6 196
• 10mM β-2-glycerophosphate = 43.2mg
• 5mM MgCl2 (203.3 g/mol) = 20.33mg
• 50mM NaFl (42 g/mol) = 42mg
• 1mM EDTA (372.2g/mol) = 7.4mg
• 0.1mM KCl (10mM stock) = 20uL
Split buffer into 2 x 10mL aliquots, 1 x 10mL with 1.5% BSA and 1 x 10mL BSA free.
Therefore 10mL buffer with 1.5% BSA = 150mg BSA added.
Note: A minimum of 70mg of muscle tissue is required to reliably and repeatedly extract
myofibrillar and mitochondrial (combining IMF and SS mitochondria) protein fractions using
a [13
C6] phenylalanine tracer.
Figure 6.1 Protein isolation protocol. Procedure for separating myofibrillar, IMF and SS mitochondrial, collagen and sarcoplasmic protein fractions
Dounce homogenization of muscle on ice
1. Add 10 µl /mg cold homogenization buffer (+BSA) to sample in the 1.5mL eppendorf. Use Teflon pestle whilst sample is still frozen to homogenize until
majority of tissue suspended.
2. Spin tube at 1000 g for 10 min @ 4ºC
3. Transfer supernatant with pipette (being careful not to pipette any of the pellet) to another eppendorf labelled SS MITO
1.5ml eppendorf tube
1. Add ~400 µl of homogenization buffer (+BSA) to the pellet
2. Homogenize with Teflon pestle. Transfer all to glass
Dounce homogenizer.
3. Homogenize in Dounce tube with glass pestle leaving no
large amts of tissue (TIGHT pestle is best).
4. Transfer to new eppendorf labelled MYO
5. Add 100µl more buffer (+BSA) to Dounce to wash extra
muscle in and also transfer to MYOFIB tube.
6. Spin at 1000 g for 10 min @ 4ºC
7. Pipette off supernatant to new 1.5mL eppendorf labelled
IMF MITO
SS and IMF MITO tubes
1. Spin tubes at 10,000 g for 10 min @ 4ºC
2. Remove supernatants and put in one 1.5mL eppendorf labelled SARC being careful not to
disturb mitochondrial pellet
MYO and COLLAGEN pellet
1. Add 500 µl homogenizing buffer (-BSA) and vortex to
fully suspend. Spin at 1000g for 5 min @ 4ºC.
2. Remove and discard supernatant. Repeat (to remove
BSA protein from myofibrillar and collagen pellet)
3. To the remaining pellet add 1 mL 0.3 M NaOH. Vortex
thoroughly and heat @ 37 ºC for 30 mins.
4. Transfer supernatant in glass vials for IRMS.
5. Add 500 µl 0.3 M NaOH and vortex to fully suspend
then heat @ 37 ºC for 10 mins
6. Remove supernatant and add to glass vial. Pellet contains
COLLAGEN.
SS and IMF MITO pellets
1. Add 500 µl buffer (-BSA). Vortex to fully suspend.
Spin at 10,000g for 5 min @ 4 ºC.
2. Remove and discard supernatant
3. Repeat steps 1 and 2.
4. Add 500 µl EtOH (95%). Vortex and spin @ 10,000g
for 10 min @ 4 ºC.
5. Remove and discard EtOH
6. Add 500µL 0.05 M HCL to pellet and break up using
pipette.
7. Add re-suspended pellet to new glass tube
8. Repeat steps 6 and 6
9. Add 1mL Dowex resin and hydrolyze overnight @ 110
ºC
MYO tube (Myofibrillar protein recovery)
1. To the supernatant add 1000 µl of 1 M PCA.
2. Spin tube at 1000 g for ~10 min @ 4ºC. Place vials (without caps) in centrifuge cups using
tweezers.
3. Remove and discard supernatant
4. Add 500 µl EtOH. Spin at 1000g for 10 mins @ 4ºC. Remove and discard EtOH.
5. Add 500µL 0.05 M HCL to pellet and break up using pipette
6. Repeat steps 6-9 of SS and IMF MITO pellets (see above)
SARC tube
1. Add 500 µl of 1 M PCA.
Vortex to suspend. Spin at
10,000g for 5 min @ 4ºC
(precipitate the AA’s).
2. Remove and discard
supernatant
3. Add 500 µl EtOH (95%).
Vortex quickly do not re-
suspend pellet. Spin at 10,000g
for 5 min @ 4 ºC.
4. Repeat steps 6-9 of SS and
IMF MITO pellets (see right)
Chapter 6 198
6.1.4 Analysis of [13C6] phenylalanine enrichment by gas chromatography-combustion-isotope
ratio mass spectrometry
Establish: 13
C6 phenylalanine in mitochondrial and myofibrillar fractions.
Rate (°C/min): 10°C/min 1.0°C/min 20°C/min
Temp (°C): 110°C 235°C 245°C 280
Time (min): 1.0’ 3.0’ 1.0’ 10.0’
• Column: Agilent technologies DB 1701 (length 30 m, I.D. 0.25 mm, film 1µm).
• RT: 33-34 min (run standard prior to every run. RT may change as column ages).
Inlet method
Base Temp: On
Base Temp: 250°C
Mode: Splitless
Split flow (ml/min): 50
Splitless time (min): 1
Surge pressure: Off
Surge pressure (kPa): 3
Surge duration (min): 0
Constant purge: On
Stop purge at (min): 0
Autosampler method
Sample volume: 1 ul
Air volume: 0.5 ul
Injection delay: 2 s
Pullout delay: 1 s
Injection speed: 100 ul/s
Pull-ups pumps: 5
Pull-ups volume: 5 ul
Chapter 6 199
Pull-ups delay: 0 s
Pre-injection washes: 1
Pre-injection volume: 5 ul
Post-injection washes: 3
Post-injection volume: 8 ul
6.1.5 Citrate synthase activity assay
A citrate synthase (CS) activity assay was used as a measure of the mitochondrial
content of each fraction. In short, the different fractions obtained from the muscle
homogenization were re-suspended in the homogenization buffer (described above, minus
BSA), containing 0.1% Triton-X to solubilize the mitochondrial inner and outer membranes.
A Bradford assay was performed in order to determine protein yield. An equal amount of
protein from each fraction was tested for CS activity. Our aim was to achieve a low level of
CS activity in the sarcoplasmic and mixed muscle fractions and a high level of CS activity in
the mitochondrial fractions. The protocol for the CS activity assay was as follows; 1mL of
0.1 M Tris buffer, pH 8.0, heated to 37°C, was added to a cuvette containing 10 µl of 10mM
DTNB in 0.1M Tris buffer and 2 µl of 30 mM acetyl-CoA in water. To this, a volume of the
muscle homogenate was added to obtain 12 µg of protein, which was then tapped gently by
hand to mix. The spectrophotometer (Thermo, Helios Lambda) was zeroed and 10 µl of 50
mM oxaloacetic acid in 0.1M Tris buffer were added to start the reaction. Absorbance
were
recorded at 412 nm every 30 s for 5 min. The premise of this assay is that the faster the
absorbance changes the greater the CS activity. CS activity assay was performed on human
skeletal muscle tissue (vastus lateralis) obtained during pilot testing.
Chapter 6 200
6.1.6 Western blots
We tested for the presence of myofibrillar and mitochondrial proteins in each fraction
using gel electrophoresis. In brief, Bradford assays were used to determine the protein
concentration of each fraction, after which samples were standardized to 1 mg/ml by dilution
with Laemmli loading buffer in. Fifteen µg of protein/lane was loaded onto Criterion XT
Bis–Tris 12% SDS-PAGE gels (Bio-Rad, Hemel Hempstead, UK) for electrophoresis at 200
V for 60 min. Gels were equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, 10%
methanol) for 30 min before proteins were electroblotted onto 0.2 lm PVDF membranes (Bio-
Rad) at 100 V for 30 min. After blocking with 5% low-fat milk in TBS-T (Tris Buffered
Saline and 0.1% Tween-20; both Sigma-Aldrich, Poole, UK) for 1 h, membranes were
rotated overnight with primary antibody against the aforementioned targets at a concentration
Figure 6.2 Citrate synthase activity assay. Change in absorbance of 12µg of mixed and
sarcoplasmic protein (low activity) and intermyofibrillar and sub-sarcolemmal mitochondrial
protein (high activity) obtained from human skeletal muscle tissue.
Time (min)
0 50 100 150 200 250 300
CS
activity (
Ab
so
rban
ce
)
0.00
0.05
0.10
0.15
0.20
0.25
0.30 SS MITO
IMF MITO
SARC
MIXED
Chapter 6 201
of 1:2,000 at 4°C. Membranes were washed (3 x 5 min) with TBS-T and incubated for 1 h at
room temperature with HRP-conjugated anti-rabbit secondary antibody (New England
Biolabs, UK), before further washing (3 x 5 min) with TBS-T and incubation for 5 min with
ECL reagents (enhanced chemiluminescence kit, Immunstar; Bio-Rad). Blots were imaged
visually inspected for the presence of slow myosin heavy chain (MHCI; DSHB A4.951) and
cytochrome C oxidase (COX; Cell Signalling 4280) protein using the Chemidoc XRS system
(Bio-Rad, Hemel Hempstead, UK). A positive test would reveal the presence of slow MHC
isoforms in the myofibrillar fraction but not in the mitochondrial fractions. Likewise, a
positive test would reveal the presence of COX in the mitochondrial, but not myofibrillar
fractions.
Figure 6.3 Western blot analysis of isolated protein fraction purity. (A) Total slow myosin
heavy chain protein content in HeLa control (CTRL), sarcoplasmic (SARC), rat myofibrillar
(MYOR), human myofibrillar (MYOH), intermyofibrillar mitochondria (IMF MITO) fractions;
(B) Total Cytochrome C Oxidase protein content in SARC, IMF MITO and MYOH fractions.
NB: For blot (A) IMF MITO was analyzed in triplicate, for blot (B) all fractions were analyzed
in triplicate.
CTRL SARC MYOR R MYOH CTRL IMF MITO (A)
14kDa
200kDa
SARC IMF MITO MYOH (B)
Chapter 6 202
6.2 Method of plasma glucose enrichment
6.1.1 Reagent preparation
• Methanol
• Chloroform
• Distilled water with pH 2 (use HCL, about 4ml 1M HCL in 500ml water)
• Butaneboronic acid
• Acetic anhydride
• Ethylacetate
• Pyridine
6.1.2 Protocol for deproteinisation and derivitization of samples
• 150 µL plasma or standard in glass screw-cap tube.
• + 150 µL of distilled water PH 2.
• 1 min vortex (plate shaker).
• +3 mL methanol:chloroform (2.3:1) (500 mL = 348:152).
• 5 min vortex (plate shaker).
• 10 min on ice.
• 15 min centrifugation at 4ºC, 3500 rpm.
Pipette top clear layer into a new glass screw-cap tube (use glass pipette)
• + 2 mL chloroform.
• + 1 mL distilled water pH 2.
• 15 min vortex (plate shaker).
• 15 min centrifugation at 4ºC, 3500 rpm.
Pipette top clear layer into a new glass screw-cap tube (using glass pipette)
• Dry for ~ 3 hours under nitrogen at 40ºC.
• The above plasma sample or 30 µL standard + 150 µL Butaneboronic acid (10 mg /
1mL pyridine).
• 15 min vortex (plate shaker).
• 30 min incubate at 95ºC.
Chapter 6 203
• +150 µL acetic anhydride.
• 90 min vortex (plate shaker).
Dry under nitrogen at 50ºC (check every 15 min).
Prepare sample for GC-MS
• +150 µL ethylacetate.
• 10 min vortex (plate shaker).
Transfer in glass vial to GC-MS
6.1.3 Analysis of [U-13C] and 6, 6-[2H2] glucose enrichment by gas chromatography- mass
spectrometry
Establish: U-13
C and 2H2 glucose in plasma.
Rate (°C/min): 4°C/min 50°C/min
Temp (°C): 135°C 215°C 250°C
Time (min): 1.0’ 2.0’ 2.0’
• Column: HP-5MS (30m length, 0.25mm I.D., 0.25um film)
Front inlet: Split injection, split ratio: 50:1
Flow rate: 2ml /min
Solvent delay: 1.5 min
6.3 References
1. Bohe J, Low JF, Wolfe RR, and Rennie MJ. Latency and duration of stimulation of
human muscle protein synthesis during continuous infusion of amino acids. J Physiol 532:
575-579, 2001.
2. Hoppeler H. Exercise-induced ultrastructural changes in skeletal muscle. Int J Sports
Med 7: 187-204, 1986.
Chapter 6 204
3. Hoppeler H, Luthi P, Claassen H, Weibel ER, and Howald H. The ultrastructure
of the normal human skeletal muscle. A morphometric analysis on untrained men, women
and well-trained orienteers. Pflugers Arch 344: 217-232, 1973.
4. Kiessling KH, Pilstrom L, Karlsson J, and Piehl K. Mitochondrial volume in
skeletal muscle from young and old physically untrained and trained healthy men and from
alcoholics. Clin Sci 44: 547-554, 1973.
5. Martin TP. Predictable adaptations by skeletal muscle mitochondria to different
exercise training workloads. Comp Biochem Physiol B 88: 273-276, 1987.
6. Rooyackers OE, Adey DB, Ades PA, and Nair KS. Effect of age on in vivo rates of
mitochondrial protein synthesis in human skeletal muscle. Proc Natl Acad Sci U S A 93:
15364-15369, 1996.
7. Wilkinson SB, Phillips SM, Atherton PJ, Patel R, Yarasheski KE, Tarnopolsky
MA, and Rennie MJ. Differential effects of resistance and endurance exercise in the fed
state on signalling molecule phosphorylation and protein synthesis in human muscle. J
Physiol 586: 3701-3717, 2008.